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Category Archives: Genetic Engineering

Principles of Genetic Engineering – PMC – National Center for …

Posted: March 28, 2024 at 2:40 am

Genes (Basel). 2020 Mar; 11(3): 291.

1Biomedical Research Core Facilities, Vector Core, University of Michigan, Ann Arbor, MI 48109, USA; ude.hcimu@tnaginal (T.M.L.); ude.hcimu@hgnohc (H.C.K.)

2Department of Internal Medicine, Division of Rheumatology, University of Michigan, Ann Arbor, MI 48109, USA

1Biomedical Research Core Facilities, Vector Core, University of Michigan, Ann Arbor, MI 48109, USA; ude.hcimu@tnaginal (T.M.L.); ude.hcimu@hgnohc (H.C.K.)

3Department of Human Genetics, University of Michigan, Ann Arbor, MI 48109, USA

4Biomedical Research Core Facilities, Transgenic Animal Model Core, University of Michigan, Ann Arbor, MI 48109, USA

5Department of Internal Medicine, Division of Genetic Medicine, University of Michigan, Ann Arbor, MI 48109, USA

2Department of Internal Medicine, Division of Rheumatology, University of Michigan, Ann Arbor, MI 48109, USA

3Department of Human Genetics, University of Michigan, Ann Arbor, MI 48109, USA

4Biomedical Research Core Facilities, Transgenic Animal Model Core, University of Michigan, Ann Arbor, MI 48109, USA

5Department of Internal Medicine, Division of Genetic Medicine, University of Michigan, Ann Arbor, MI 48109, USA

These authors contributed to the work equally.

Received 2019 Dec 31; Accepted 2020 Mar 6.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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Historic Overview of Genetic Engineering Technologies for Human Gene …

Posted: March 28, 2024 at 2:40 am

Abstract

The concepts of gene therapy were initially introduced during the 1960s. Since the early 1990s, more than 1900 clinical trials have been conducted for the treatment of genetic diseases and cancers mainly using viral vectors. Although a variety of methods have also been performed for the treatment of malignant gliomas, it has been difficult to target invasive glioma cells. To overcome this problem, immortalized neural stem cell (NSC) and a nonlytic, amphotropic retroviral replicating vector (RRV) have attracted attention for gene delivery to invasive glioma. Recently, genome editing technology targeting insertions at site-specific locations has advanced; in particular, the clustered regularly interspaced palindromic repeats/CRISPR-associated-9 (CRISPR/Cas9) has been developed. Since 2015, more than 30 clinical trials have been conducted using genome editing technologies, and the results have shown the potential to achieve positive patient outcomes. Gene therapy using CRISPR technologies for the treatment of a wide range of diseases is expected to continuously advance well into the future.

Keywords: gene therapy, genome editing, ZFN, TALEN, CRISPR/Cas9

Gene therapy is a therapeutic strategy using genetic engineering techniques to treat various diseases.1,2) In the early 1960s, gene therapy first progressed with the development of recombinant DNA (rDNA) technology,1) and was further developed using various genetic engineering tools, such as viral vectors.35) More than 1900 clinical trials have been conducted with gene therapeutic approaches since the early 1990s. In these procedures, DNA is randomly inserted into the host genome using conventional genetic engineering tools. In the 2000s, genome editing tootls, including zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the recently established clustered regularly interspaced palindromic repeats/CRISPR-associated-9 (CRISPR/Cas9) technologies, were developed, which induce genome modifications at specific target sites.5) Genome editing tools are efficient for intentional genetic engineering, which has led to the development of novel treatment strategies for a wide range of diseases, such as genetic diseases and cancers. Therefore, gene therapy has again became a major focus of medical research. However, because gene therapy involves changing the genetic background, it raises important ethical concerns. In this article, we review the brief history of gene therapy and the development of genetic engineering technologies.

In 1968, the initial proof-of-concept of virus- mediated gene transfer was made by Rogers et al.6) who showed that foreign genetic material could be transferred into cells by viruses. In the first human gene therapy experiment, Shope papilloma virus was transduced into two patients with genetic arginase deficiency, because Rogers et al. hypothesized that the Shope papilloma virus genome contained a gene that encodes arginase. However, this gene therapy produced little improvement in the arginase levels in the patients.7) Sequencing of the Shope papilloma virus genome revealed that the virus genome did not contain an arginase gene.7)

This experiment prompted public concerns about the risks and ethical issues of gene therapy. In 1972, Friedman et al.8) proposed ethical standards for the clinical application of gene therapy to prevent premature application in human. However, in 1980, genetic engineering was unethically performed in patients with thalassemia without the approval of the institutional review board.9) The patients bone marrow cells were harvested and returned into their bone marrow after transduction with the plasmid DNA containing an integrated b-globin gene.9) This treatment showed no effects, and the experiments were regarded as morally dubious. The gene therapy report of the President's Commission in the United States, Splicing Life, emphasized the distinction between somatic and germline genome editing in humans, and between medical treatment and non-medical enhancement.10) An altered gene inserted into sperm or egg cells (germ cells) would lead to changes not only in the individual receiving the treatment but also in their future offspring. Interventions aimed at enhancing normal people also are problematic because they might lead to attempts to make perfect human beings.

In 1980, only nonviral methods, such as microinjection and calcium-phosphate precipitation, were used for gene delivery. Nonviral methods showed some advantages compared with viral methods, such as large-scale production and low host immunogenicity. However, nonviral methods yielded lower levels of transfection and gene expression, resulting in limited therapeutic efficacy.11) In 1989, the rDNA Advisory Committee of the National Institutes of Health proposed the first guidelines for the clinical trials of gene therapy. In 1990, retroviral infection, which is highly dependent on host cell cycle status, was first performed for the transduction of the neomycin resistance marker gene into tumor-infiltrating lymphocytes that were obtained from patients with metastatic melanoma.3,4) Then, the lymphocytes were cultured in vitro and returned to the patients bodies.3,4) The first Food and Drug Administration (FDA)- approved gene therapy using a retroviral vector was performed by Anderson et al. in 1990; the adenosine deaminase (ADA) gene was transduced into the white blood cells of a patient with ADA deficiency, resulting in temporary improvements in her immunity.2,12)

A recombinant adenoviral (AV) vector was developed after advances in the use of the retroviral vector. In 1999, a clinical trial was performed for ornithine transcarbamylase (OTC) deficiency. A ubiquitous DNA AV vector (Ad5) containing the OTC gene was delivered into the patient. Four days after administration, the patient died from multiple organ failure that was caused by a cytokine storm.13,14) In 1999, of the 20 patients enrolled in two trials for severe combined immunodeficiency (SCID)-X1, T-cell leukemia was observed in five patients at 25.5 years after the treatment. Hematopoietic stem cells with a conventional, amphotropic, murine leukemia virus-based vector and a gibbon-ape leukemia virus-pseudotyped retrovirus were used for gene transduction in those trials.15,16) Although four patients fully recovered after the treatment, one patient died15,16) because oncogene activation was mediated by viral insertion.15,16)

Viral vectors continued to be crucial components in the manufacture of cell and gene therapy. Adeno- associated viral (AAV) vectors were applied for many genetic diseases including Lebers Congenital Amaurosis (LCA), and reverse lipoprotein lipase deficiency (LPLD). In 2008, remarkable success was reported for LCA type II in phase I/II clinical trials.17) LCA is a rare hereditary retinal degeneration disorder caused by mutations in the RPE65 gene (Retinoid Isomerohydrolase RPE65), which is highly expressed in the retinal pigment epithelium and encodes retinoid isomerase.17) These trials confirm that RPE65 could be delivered into retinal pigment epithelial cells using recombinant AAV2/2 vectors, resulting in clinical benefits without adverse events.17) Recently, the FDA approved voretigene neparvovec-rzyl (Luxturna, Spark Therapeutics, Philadelphia, PA, USA) for patients with LCA type II. Alipogene tiparvovec Glybera (uniQure, Lexington, MA, USA) is the first gene-therapy-based drug to reverse LPLD to be approved in Europe in 2012. The AAV1 vector delivers an intact LPL gene to the muscle cells.18) To date, more than 200 clinical trials have been performed using AAV vectors for several genetic diseases, including spinal muscular atrophy,19) retinal dystrophy,20) and hemophilia.21)

Retrovirus is still one of the mainstays of gene therapeutic approaches. Strimvelis (GlaxoSmithKline, London, UK) is an FDA-approved drug consisting of an autologous CD34 (+)-enriched cell population that includes a gammaretrovirus containing the ADA gene that was used as the first ex-vivo stem cell gene therapy in patients with SCID because of ADA deficiency.22) Subsequently, retroviral vectors were often used for other genetic diseases, including X-SCID.23)

Lentivirus belongs to a family of viruses that are responsible for diseases, such as aquired immunodeficiency syndrome caused by the human immunodeficiency virus (HIV) that causes infection by inserting DNA into the genome of their host cells.24) The lentivirus can infect non-dividing cells; therefore, it has a wider range of potential applications. Successful treatment of the patients with X-linked adrenoleukodystrophy was demonstrated using a lentiviral vector with the deficient peroxisomal adenosine triphosphatebinding cassette D1.25) Despite the use of a lentiviral vector with an internal viral long terminal repeat, no oncogene activation was observed.25)

A timeline showing the history of scientific progress in gene therapy is highlighted in .

History of gene therapy

A variety of studies were performed to apply gene therapy to malignant tumors. The concept of gene therapy for tumors is different from that for genetic diseases, in which new genes are added to a patient's cells to replace missing or malfunctioning genes. In malignant tumors, the breakthrough in gene therapeutic strategy involved designing suicide gene therapy,26) which was first applied for malignant glioma in 1992.26,27) The first clinical study was performed on 15 patients with malignant gliomas by Ram et al (phase I/II).27) Stereotactic intratumoral injections of murine fibroblasts producing a replication-deficient retrovirus vector with a suicide gene (herpes simplex virus-thymidine kinase [HSV-TK]) achieved anti-tumor activity in four patients through bystander killing effects.27) Subsequently, various types of therapeutic genes have been used to treat malignant glioma. Suicide genes (cytosine deaminase [CD]), genes for immunomodulatory cytokines (interferon [IFN]-, interleukin [IL]-12, granulocyte- macrophage colony-stimulating factor [GM-CSF]), and genes for reprogramming (p53, and phosphatase and tensin homolog deleted from chromosome [PTEN]) have been applied to the treatment of malignant glioma using viral vectors.28,29)

Recently, a nonlytic, amphotropic retroviral replicating vector (RRV) and immortalized human neural stem cell (NSC) line were used for gene delivery to invasive glioma.3032) In 2012, a nonlytic, amphotropic RRV called Toca 511 was developed for the delivery of a suicide gene (CD) to tumors.32) A tumor-selective Toca 511 combined with a prodrug (Toca FC) was evaluated in patients with recurrent high-grade glioma in phase I clinical trial.30) The complete response rate was 11.3% in 53 patients.30) In addition, the sub-analysis of this clinical trial revealed that the objective response was 21.7% in the 23-patient phase III eligible subgroup.33) However, in the recent phase III trial, treatment with Toca 511 and Toca FC did not improve overall survival compared with standard therapy in patients with recurrent high-grade glioma. A further combinational treatment strategy using programmed cell-death ligand 1 (PD-L1) checkpoint blockade delivered by TOCA-511 was evaluated in experimental models, which may lead to future clinical application.34) Since 2010, intracranial administration of allogeneic NSCs containing CD gene (HB1.F3. CD) has been performed by a team at City of Hope. Autopsy specimens indicate the HB1.F3. CD migrates toward invaded tumor areas, suggesting a high tumor-trophic migratory capacity of NSCs.31) No severe toxicities were observed in the trial. Generally, it is difficult to obtain NSCs derived from human embryonic or fetal tissue. The use of human embryos for research on embryonic stem cells is ethically controversial because it involves the destruction of human embryos, and the use of fetal tissue associated with abortion also raises ethical considerations.35) Recently, the tumor-trophic migratory activity of NSCs derived from human-induced pluripotent stem cells (hiPSCs) was shown using organotypic brain slice culture.36) Moreover, hiPSC-derived NSCs with the HSV-TK suicide gene system demonstrated considerable therapeutic potential for the treatment of experimental glioma models.36) Furthermore, iPSCs have the ability to overcome ethical and practical issues of NSCs in clinical application.

Genetic engineering technologies using viral vectors to randomly insert therapeutic genes into a host genome raised concerns about insertional mutagenesis and oncogene activation. Therefore, new technology to intentionally insert genes at site-specific locations was needed. Genome editing is a genetic engineering method that uses nucleases or molecular scissors to intentionally introduce alterations into the genome of living organisms.6) As of 2015, three types of engineered nucleases have been used: ZFNs, TALENs, and CRISPR/Cas ().6)

Characteristics of genome editing technologies

ZFNs are fusions of the nonspecific DNA cleavage domain of the Fok I restriction endonuclease and zinc-finger proteins that lead to DNA double-strand breaks (DSBs). Zinc-finger domains recognize a trinucleotide DNA sequence (). However, design and selection of zinc-finber arrays is difficult and time-consuming.37)

Genome editing tools. Three types of genome editing tools including ZFNs, TALENs, and CRISPR/Cas9 are shown. ZFNs are hybrid proteins using zinc-finger arrays and the catalytic domain of FokI endonuclease. TALENs are hybrid proteins containing the TAL effector backbone and the catalytic domain of FokI endonuclease. The CRISPR/Cas9 system is composed of Cas9 endonuclease and sgRNA. Cas9: CRISPR-associated-9, CRISPR: clustered regularly interspaced palindromic repeats, sgRNA: single-guide RNA, TALENs: transcription activator-like effector nucleases, ZFNs: zinc-finger nucleases.

TALENs are fusions of the Fok I cleavage domain and DNA-binding domains derived from TALE proteins. TALEs have multiple 3335 amino acid repeat domains that recognizes a single base pair, leading to the targeted DSBs, similar to ZFNs ().38)

The CRISPR/Cas9 system consists of Cas9 nuclease and two RNAs (CRISPR RNA [crRNA] and trans- activating CRISPR RNA [tracrRNA]).39) The crRNA/tracrRNA complex (gRNA) induces the Cas9 nuclease and cleaves DNA upstream of a protospacer-adjacent motif (PAM, 5-NGG-3 for S. pyogenes) ().40) Currently, Cas9 from S. pyogenes (SpCas9) is the most popular tool for genome editing.40)

Several studies have demonstrated the off-target effects of Cas9/gRNA complexes.41) It is important to select unique target sites without closely homologous sequences, resulting in minimum off-target effects.42) Additionally, other CRISPR/Cas9 gene editing tools were developed to mitigate off-target effects, including gRNA modifications (slightly truncated gRNAs with shorter regions of target complementarity <20 nucleotides)43) and SpCas9 variants, such as Cas9 paired nickases (a Cas9 nickase mutant or dimeric Cas9 proteins combined with pairs of gRNAs).44) The type I CRISPR-mediated distinct DNA cleavage (CRISPR/Cas3 system) was developed recently in Japan to decrease the risk of off-target effets. Cas3 triggered long-range deletions upstream of the PAM (5'-ARG).45)

A confirmatory screening of off-target effects is necessary for ensuring the safe application of genome editing technologies.46) Although off-target mutations in the genome, including the noncoding region, can be evaluated using whole genome sequencing, this method is expensive and time-consuming. With the development of unbiased genome-wide cell-based methods, GUIDE-seq (genome-wide, unbiased identification of DSBs enabled by sequencing)47) and BLESS (direct in situ breaks labeling, enrichment on streptavidin; next-generation sequencing)48) were developed to detect off-target cleavage sites, and these methods do not require high sequencing read counts.

HIV-resistant T cells were established by ZFN- mediated disruption of the C-C chemokine receptor (CCR) 5 coreceptor for HIV-I, which is being evaluated as an ex- vivo modification in early-stage clinical trials.49,50) Disruption of CCR5 using ZFNs was the first-in-human application of a genome editing tool. Regarding hematologic disorders, since 2016, clinical trials have attempted the knock-in of the factor IX gene using AAV/ZFN-mediated genome editing approach for patients with hemophilia B.51)

In addition to these promising ongoing clinical trials for genetic diseases, CRISPR/Cas9 and TALEN technologies have improved the effect of cancer immunotherapy using genome-engineered T cells. Engineered T cells express synthetic receptors (chimeric antigen receptors, CARs) that can recognize epitopes on tumor cells. The FDA approved two CD19-targeting CAR-T-cell products for B-cell acute lymphoblastic leukemia and diffuse large B-cell lymphoma.52,53) Engineered CARs target many other antigens of blood cancers, including CD30 in Hodgkin's lymphoma as well as CD33, CD123, and FLT3 of acute myeloid leukemia.54) Recent research has shown that Cas9-mediated PD-1 disruption in the CAR-T cells improved the anti-tumor effect observed in in- vitro and in- vivo experimental models, leading to the performance of a clinical trial.55,56) All other ongoing clinical trials using genome-editing technologies are highlighted in .

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20.3: Genetic Engineering – Biology LibreTexts

Posted: December 10, 2023 at 2:33 am

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

OpenStax, Biology. OpenStax CNX. May 27, 2016 http://cnx.org/contents/s8Hh0oOc@9.10:8CA_YwJq@3/Cloning-and-Genetic-Engineerin

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

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20.3: Genetic Engineering - Biology LibreTexts

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Genetically modified organism – Wikipedia

Posted: November 16, 2023 at 2:51 pm

Organisms whose genetic material has been altered using genetic engineering methods

A genetically modified organism (GMO) is any organism whose genetic material has been altered using genetic engineering techniques. The exact definition of a genetically modified organism and what constitutes genetic engineering varies, with the most common being an organism altered in a way that "does not occur naturally by mating and/or natural recombination".[1] A wide variety of organisms have been genetically modified (GM), from animals to plants and microorganisms. Genes have been transferred within the same species, across species (creating transgenic organisms), and even across kingdoms. New genes can be introduced, or endogenous genes can be enhanced, altered, or knocked out.

Creating a genetically modified organism is a multi-step process. Genetic engineers must isolate the gene they wish to insert into the host organism and combine it with other genetic elements, including a promoter and terminator region and often a selectable marker. A number of techniques are available for inserting the isolated gene into the host genome. Recent advancements using genome editing techniques, notably CRISPR, have made the production of GMOs much simpler. Herbert Boyer and Stanley Cohen made the first genetically modified organism in 1973, a bacterium resistant to the antibiotic kanamycin. The first genetically modified animal, a mouse, was created in 1974 by Rudolf Jaenisch, and the first plant was produced in 1983. In 1994, the Flavr Savr tomato was released, the first commercialized genetically modified food. The first genetically modified animal to be commercialized was the GloFish (2003) and the first genetically modified animal to be approved for food use was the AquAdvantage salmon in 2015.

Bacteria are the easiest organisms to engineer and have been used for research, food production, industrial protein purification (including drugs), agriculture, and art. There is potential to use them for environmental purposes or as medicine. Fungi have been engineered with much the same goals. Viruses play an important role as vectors for inserting genetic information into other organisms. This use is especially relevant to human gene therapy. There are proposals to remove the virulent genes from viruses to create vaccines. Plants have been engineered for scientific research, to create new colors in plants, deliver vaccines, and to create enhanced crops. Genetically modified crops are publicly the most controversial GMOs, in spite of having the most human health and environmental benefits.[2] The majority are engineered for herbicide tolerance or insect resistance. Golden rice has been engineered with three genes that increase its nutritional value. Other prospects for GM crops are as bioreactors for the production of biopharmaceuticals, biofuels, or medicines.

Animals are generally much harder to transform and the vast majority are still at the research stage. Mammals are the best model organisms for humans, making ones genetically engineered to resemble serious human diseases important to the discovery and development of treatments. Human proteins expressed in mammals are more likely to be similar to their natural counterparts than those expressed in plants or microorganisms. Livestock is modified with the intention of improving economically important traits such as growth rate, quality of meat, milk composition, disease resistance, and survival. Genetically modified fish are used for scientific research, as pets, and as a food source. Genetic engineering has been proposed as a way to control mosquitos, a vector for many deadly diseases. Although human gene therapy is still relatively new, it has been used to treat genetic disorders such as severe combined immunodeficiency and Leber's congenital amaurosis.

Many objections have been raised over the development of GMOs, particularly their commercialization. Many of these involve GM crops and whether food produced from them is safe and what impact growing them will have on the environment. Other concerns are the objectivity and rigor of regulatory authorities, contamination of non-genetically modified food, control of the food supply, patenting of life, and the use of intellectual property rights. Although there is a scientific consensus that currently available food derived from GM crops poses no greater risk to human health than conventional food, GM food safety is a leading issue with critics. Gene flow, impact on non-target organisms, and escape are the major environmental concerns. Countries have adopted regulatory measures to deal with these concerns. There are differences in the regulation for the release of GMOs between countries, with some of the most marked differences occurring between the US and Europe. Key issues concerning regulators include whether GM food should be labeled and the status of gene-edited organisms.

The definition of a genetically modified organism (GMO) is not clear and varies widely between countries, international bodies, and other communities. At its broadest, the definition of a GMO can include anything that has had its genes altered, including by nature.[3][4] Taking a less broad view, it can encompass every organism that has had its genes altered by humans, which would include all crops and livestock. In 1993, the Encyclopedia Britannica defined genetic engineering as "any of a wide range of techniques... among them artificial insemination, in vitro fertilization (e.g., 'test-tube' babies), sperm banks, cloning, and gene manipulation."[5] The European Union (EU) included a similarly broad definition in early reviews, specifically mentioning GMOs being produced by "selective breeding and other means of artificial selection"[6] These definitions were promptly adjusted with a number of exceptions added as the result of pressure from scientific and farming communities, as well as developments in science. The EU definition later excluded traditional breeding, in vitro fertilization, induction of polyploidy, mutation breeding, and cell fusion techniques that do not use recombinant nucleic acids or a genetically modified organism in the process.[7][8][9]

Another approach was the definition provided by the Food and Agriculture Organization, the World Health Organization, and the European Commission, stating that the organisms must be altered in a way that does "not occur naturally by mating and/or natural recombination".[10][11][12] Progress in science, such as the discovery of horizontal gene transfer being a relatively common natural phenomenon, further added to the confusion on what "occurs naturally", which led to further adjustments and exceptions.[13] There are examples of crops that fit this definition, but are not normally considered GMOs.[14] For example, the grain crop triticale was fully developed in a laboratory in 1930 using various techniques to alter its genome.[15]

Genetically engineered organism (GEO) can be considered a more precise term compared to GMO when describing organisms' genomes that have been directly manipulated with biotechnology.[16][8] The Cartagena Protocol on Biosafety used the synonym living modified organism (LMO) in 2000 and defined it as "any living organism that possesses a novel combination of genetic material obtained through the use of modern biotechnology."[17] Modern biotechnology is further defined as "In vitro nucleic acid techniques, including recombinant deoxyribonucleic acid (DNA) and direct injection of nucleic acid into cells or organelles, or fusion of cells beyond the taxonomic family."[18]

The term GMO originally was not typically used by scientists to describe genetically engineered organisms until after usage of GMO became common in popular media.[19] The United States Department of Agriculture (USDA) considers GMOs to be plants or animals with heritable changes introduced by genetic engineering or traditional methods, while GEO specifically refers to organisms with genes introduced, eliminated, or rearranged using molecular biology, particularly recombinant DNA techniques, such as transgenesis.[20]

The definitions focus on the process more than the product, which means there could be GMOS and non-GMOs with very similar genotypes and phenotypes.[21][22] This has led scientists to label it as a scientifically meaningless category,[23] saying that it is impossible to group all the different types of GMOs under one common definition.[24] It has also caused issues for organic institutions and groups looking to ban GMOs.[25][26] It also poses problems as new processes are developed. The current definitions came in before genome editing became popular and there is some confusion as to whether they are GMOs. The EU has adjudged that they are[27] changing their GMO definition to include "organisms obtained by mutagenesis", but has excluded them from regulation based on their "long safety record" and that they have been "conventionally been used in a number of applications".[9] In contrast the USDA has ruled that gene edited organisms are not considered GMOs.[28]

Even greater inconsistency and confusion is associated with various "Non-GMO" or "GMO-free" labeling schemes in food marketing, where even products such as water or salt, which do not contain any organic substances and genetic material (and thus cannot be genetically modified by definition), are being labeled to create an impression of being "more healthy".[29][30][31]

Creating a genetically modified organism (GMO) is a multi-step process. Genetic engineers must isolate the gene they wish to insert into the host organism. This gene can be taken from a cell[32] or artificially synthesized.[33] If the chosen gene or the donor organism's genome has been well studied it may already be accessible from a genetic library. The gene is then combined with other genetic elements, including a promoter and terminator region and a selectable marker.[34]

A number of techniques are available for inserting the isolated gene into the host genome. Bacteria can be induced to take up foreign DNA, usually by exposed heat shock or electroporation.[35] DNA is generally inserted into animal cells using microinjection, where it can be injected through the cell's nuclear envelope directly into the nucleus, or through the use of viral vectors.[36] In plants the DNA is often inserted using Agrobacterium-mediated recombination,[37][38] biolistics[39] or electroporation.

As only a single cell is transformed with genetic material, the organism must be regenerated from that single cell. In plants this is accomplished through tissue culture.[40][41] In animals it is necessary to ensure that the inserted DNA is present in the embryonic stem cells.[37] Further testing using PCR, Southern hybridization, and DNA sequencing is conducted to confirm that an organism contains the new gene.[42]

Traditionally the new genetic material was inserted randomly within the host genome. Gene targeting techniques, which creates double-stranded breaks and takes advantage on the cells natural homologous recombination repair systems, have been developed to target insertion to exact locations. Genome editing uses artificially engineered nucleases that create breaks at specific points. There are four families of engineered nucleases: meganucleases,[43][44] zinc finger nucleases,[45][46] transcription activator-like effector nucleases (TALENs),[47][48] and the Cas9-guideRNA system (adapted from CRISPR).[49][50] TALEN and CRISPR are the two most commonly used and each has its own advantages.[51] TALENs have greater target specificity, while CRISPR is easier to design and more efficient.[51]

Humans have domesticated plants and animals since around 12,000 BCE, using selective breeding or artificial selection (as contrasted with natural selection).[52]:25 The process of selective breeding, in which organisms with desired traits (and thus with the desired genes) are used to breed the next generation and organisms lacking the trait are not bred, is a precursor to the modern concept of genetic modification.[53]:1[54]:1 Various advancements in genetics allowed humans to directly alter the DNA and therefore genes of organisms. In 1972, Paul Berg created the first recombinant DNA molecule when he combined DNA from a monkey virus with that of the lambda virus.[55][56]

Herbert Boyer and Stanley Cohen made the first genetically modified organism in 1973.[57] They took a gene from a bacterium that provided resistance to the antibiotic kanamycin, inserted it into a plasmid and then induced other bacteria to incorporate the plasmid. The bacteria that had successfully incorporated the plasmid was then able to survive in the presence of kanamycin.[58] Boyer and Cohen expressed other genes in bacteria. This included genes from the toad Xenopus laevis in 1974, creating the first GMO expressing a gene from an organism of a different kingdom.[59]

In 1974, Rudolf Jaenisch created a transgenic mouse by introducing foreign DNA into its embryo, making it the world's first transgenic animal.[60][61] However it took another eight years before transgenic mice were developed that passed the transgene to their offspring.[62][63] Genetically modified mice were created in 1984 that carried cloned oncogenes, predisposing them to developing cancer.[64] Mice with genes removed (termed a knockout mouse) were created in 1989. The first transgenic livestock were produced in 1985[65] and the first animal to synthesize transgenic proteins in their milk were mice in 1987.[66] The mice were engineered to produce human tissue plasminogen activator, a protein involved in breaking down blood clots.[67]

In 1983, the first genetically engineered plant was developed by Michael W. Bevan, Richard B. Flavell and Mary-Dell Chilton. They infected tobacco with Agrobacterium transformed with an antibiotic resistance gene and through tissue culture techniques were able to grow a new plant containing the resistance gene.[68] The gene gun was invented in 1987, allowing transformation of plants not susceptible to Agrobacterium infection.[69] In 2000, Vitamin A-enriched golden rice was the first plant developed with increased nutrient value.[70]

In 1976, Genentech, the first genetic engineering company was founded by Herbert Boyer and Robert Swanson; a year later, the company produced a human protein (somatostatin) in E. coli. Genentech announced the production of genetically engineered human insulin in 1978.[71] The insulin produced by bacteria, branded Humulin, was approved for release by the Food and Drug Administration in 1982.[72] In 1988, the first human antibodies were produced in plants.[73] In 1987, a strain of Pseudomonas syringae became the first genetically modified organism to be released into the environment[74] when a strawberry and potato field in California were sprayed with it.[75]

The first genetically modified crop, an antibiotic-resistant tobacco plant, was produced in 1982.[76] China was the first country to commercialize transgenic plants, introducing a virus-resistant tobacco in 1992.[77] In 1994, Calgene attained approval to commercially release the Flavr Savr tomato, the first genetically modified food.[78] Also in 1994, the European Union approved tobacco engineered to be resistant to the herbicide bromoxynil, making it the first genetically engineered crop commercialized in Europe.[79] An insect resistant Potato was approved for release in the US in 1995,[80] and by 1996 approval had been granted to commercially grow 8 transgenic crops and one flower crop (carnation) in 6 countries plus the EU.[81]

In 2010, scientists at the J. Craig Venter Institute announced that they had created the first synthetic bacterial genome. They named it Synthia and it was the world's first synthetic life form.[82][83]

The first genetically modified animal to be commercialized was the GloFish, a Zebra fish with a fluorescent gene added that allows it to glow in the dark under ultraviolet light.[84] It was released to the US market in 2003.[85] In 2015, AquAdvantage salmon became the first genetically modified animal to be approved for food use.[86] Approval is for fish raised in Panama and sold in the US.[86] The salmon were transformed with a growth hormone-regulating gene from a Pacific Chinook salmon and a promoter from an ocean pout enabling it to grow year-round instead of only during spring and summer.[87]

Bacteria were the first organisms to be genetically modified in the laboratory, due to the relative ease of modifying their chromosomes.[88] This ease made them important tools for the creation of other GMOs. Genes and other genetic information from a wide range of organisms can be added to a plasmid and inserted into bacteria for storage and modification. Bacteria are cheap, easy to grow, clonal, multiply quickly and can be stored at 80C almost indefinitely. Once a gene is isolated it can be stored inside the bacteria, providing an unlimited supply for research.[89] A large number of custom plasmids make manipulating DNA extracted from bacteria relatively easy.[90]

Their ease of use has made them great tools for scientists looking to study gene function and evolution. The simplest model organisms come from bacteria, with most of our early understanding of molecular biology coming from studying Escherichia coli.[91] Scientists can easily manipulate and combine genes within the bacteria to create novel or disrupted proteins and observe the effect this has on various molecular systems. Researchers have combined the genes from bacteria and archaea, leading to insights on how these two diverged in the past.[92] In the field of synthetic biology, they have been used to test various synthetic approaches, from synthesizing genomes to creating novel nucleotides.[93][94][95]

Bacteria have been used in the production of food for a long time, and specific strains have been developed and selected for that work on an industrial scale. They can be used to produce enzymes, amino acids, flavorings, and other compounds used in food production. With the advent of genetic engineering, new genetic changes can easily be introduced into these bacteria. Most food-producing bacteria are lactic acid bacteria, and this is where the majority of research into genetically engineering food-producing bacteria has gone. The bacteria can be modified to operate more efficiently, reduce toxic byproduct production, increase output, create improved compounds, and remove unnecessary pathways.[96] Food products from genetically modified bacteria include alpha-amylase, which converts starch to simple sugars, chymosin, which clots milk protein for cheese making, and pectinesterase, which improves fruit juice clarity.[97] The majority are produced in the US and even though regulations are in place to allow production in Europe, as of 2015 no food products derived from bacteria are currently available there.[98]

Genetically modified bacteria are used to produce large amounts of proteins for industrial use. Generally the bacteria are grown to a large volume before the gene encoding the protein is activated. The bacteria are then harvested and the desired protein purified from them.[99] The high cost of extraction and purification has meant that only high value products have been produced at an industrial scale.[100] The majority of these products are human proteins for use in medicine.[101] Many of these proteins are impossible or difficult to obtain via natural methods and they are less likely to be contaminated with pathogens, making them safer.[99] The first medicinal use of GM bacteria was to produce the protein insulin to treat diabetes.[102] Other medicines produced include clotting factors to treat hemophilia,[103] human growth hormone to treat various forms of dwarfism,[104][105] interferon to treat some cancers, erythropoietin for anemic patients, and tissue plasminogen activator which dissolves blood clots.[99] Outside of medicine they have been used to produce biofuels.[106] There is interest in developing an extracellular expression system within the bacteria to reduce costs and make the production of more products economical.[100]

With a greater understanding of the role that the microbiome plays in human health, there is a potential to treat diseases by genetically altering the bacteria to, themselves, be therapeutic agents. Ideas include altering gut bacteria so they destroy harmful bacteria, or using bacteria to replace or increase deficient enzymes or proteins. One research focus is to modify Lactobacillus, bacteria that naturally provide some protection against HIV, with genes that will further enhance this protection. If the bacteria do not form colonies inside the patient, the person must repeatedly ingest the modified bacteria in order to get the required doses. Enabling the bacteria to form a colony could provide a more long-term solution, but could also raise safety concerns as interactions between bacteria and the human body are less well understood than with traditional drugs. There are concerns that horizontal gene transfer to other bacteria could have unknown effects. As of 2018 there are clinical trials underway testing the efficacy and safety of these treatments.[107]

For over a century bacteria have been used in agriculture. Crops have been inoculated with Rhizobia (and more recently Azospirillum) to increase their production or to allow them to be grown outside their original habitat. Application of Bacillus thuringiensis (Bt) and other bacteria can help protect crops from insect infestation and plant diseases. With advances in genetic engineering, these bacteria have been manipulated for increased efficiency and expanded host range. Markers have also been added to aid in tracing the spread of the bacteria. The bacteria that naturally colonize certain crops have also been modified, in some cases to express the Bt genes responsible for pest resistance. Pseudomonas strains of bacteria cause frost damage by nucleating water into ice crystals around themselves. This led to the development of ice-minus bacteria, which have the ice-forming genes removed. When applied to crops they can compete with the non-modified bacteria and confer some frost resistance.[108]

Other uses for genetically modified bacteria include bioremediation, where the bacteria are used to convert pollutants into a less toxic form. Genetic engineering can increase the levels of the enzymes used to degrade a toxin or to make the bacteria more stable under environmental conditions.[109] Bioart has also been created using genetically modified bacteria. In the 1980s artist Jon Davis and geneticist Dana Boyd converted the Germanic symbol for femininity () into binary code and then into a DNA sequence, which was then expressed in Escherichia coli.[110] This was taken a step further in 2012, when a whole book was encoded onto DNA.[111] Paintings have also been produced using bacteria transformed with fluorescent proteins.[110]

Viruses are often modified so they can be used as vectors for inserting genetic information into other organisms. This process is called transduction and if successful the recipient of the introduced DNA becomes a GMO. Different viruses have different efficiencies and capabilities. Researchers can use this to control for various factors; including the target location, insert size, and duration of gene expression. Any dangerous sequences inherent in the virus must be removed, while those that allow the gene to be delivered effectively are retained.[112]

While viral vectors can be used to insert DNA into almost any organism it is especially relevant for its potential in treating human disease. Although primarily still at trial stages,[113] there has been some successes using gene therapy to replace defective genes. This is most evident in curing patients with severe combined immunodeficiency rising from adenosine deaminase deficiency (ADA-SCID),[114] although the development of leukemia in some ADA-SCID patients[115] along with the death of Jesse Gelsinger in a 1999 trial set back the development of this approach for many years.[116] In 2009, another breakthrough was achieved when an eight-year-old boy with Leber's congenital amaurosis regained normal eyesight[116] and in 2016 GlaxoSmithKline gained approval to commercialize a gene therapy treatment for ADA-SCID.[114] As of 2018, there are a substantial number of clinical trials underway, including treatments for hemophilia, glioblastoma, chronic granulomatous disease, cystic fibrosis and various cancers.[115]

The most common virus used for gene delivery comes from adenoviruses as they can carry up to 7.5 kb of foreign DNA and infect a relatively broad range of host cells, although they have been known to elicit immune responses in the host and only provide short term expression. Other common vectors are adeno-associated viruses, which have lower toxicity and longer-term expression, but can only carry about 4kb of DNA.[115] Herpes simplex viruses make promising vectors, having a carrying capacity of over 30kb and providing long term expression, although they are less efficient at gene delivery than other vectors.[117] The best vectors for long term integration of the gene into the host genome are retroviruses, but their propensity for random integration is problematic. Lentiviruses are a part of the same family as retroviruses with the advantage of infecting both dividing and non-dividing cells, whereas retroviruses only target dividing cells. Other viruses that have been used as vectors include alphaviruses, flaviviruses, measles viruses, rhabdoviruses, Newcastle disease virus, poxviruses, and picornaviruses.[115]

Most vaccines consist of viruses that have been attenuated, disabled, weakened or killed in some way so that their virulent properties are no longer effective. Genetic engineering could theoretically be used to create viruses with the virulent genes removed. This does not affect the viruses infectivity, invokes a natural immune response and there is no chance that they will regain their virulence function, which can occur with some other vaccines. As such they are generally considered safer and more efficient than conventional vaccines, although concerns remain over non-target infection, potential side effects and horizontal gene transfer to other viruses.[118] Another potential approach is to use vectors to create novel vaccines for diseases that have no vaccines available or the vaccines that do not work effectively, such as AIDS, malaria, and tuberculosis.[119] The most effective vaccine against Tuberculosis, the Bacillus CalmetteGurin (BCG) vaccine, only provides partial protection. A modified vaccine expressing a M tuberculosis antigen is able to enhance BCG protection.[120] It has been shown to be safe to use at phase II trials, although not as effective as initially hoped.[121] Other vector-based vaccines have already been approved and many more are being developed.[119]

Another potential use of genetically modified viruses is to alter them so they can directly treat diseases. This can be through expression of protective proteins or by directly targeting infected cells. In 2004, researchers reported that a genetically modified virus that exploits the selfish behavior of cancer cells might offer an alternative way of killing tumours.[122][123] Since then, several researchers have developed genetically modified oncolytic viruses that show promise as treatments for various types of cancer.[124][125][126][127][128] In 2017, researchers genetically modified a virus to express spinach defensin proteins. The virus was injected into orange trees to combat citrus greening disease that had reduced orange production by 70% since 2005.[129]

Natural viral diseases, such as myxomatosis and rabbit hemorrhagic disease, have been used to help control pest populations. Over time the surviving pests become resistant, leading researchers to look at alternative methods. Genetically modified viruses that make the target animals infertile through immunocontraception have been created in the laboratory[130] as well as others that target the developmental stage of the animal.[131] There are concerns with using this approach regarding virus containment[130] and cross species infection.[132] Sometimes the same virus can be modified for contrasting purposes. Genetic modification of the myxoma virus has been proposed to conserve European wild rabbits in the Iberian peninsula and to help regulate them in Australia. To protect the Iberian species from viral diseases, the myxoma virus was genetically modified to immunize the rabbits, while in Australia the same myxoma virus was genetically modified to lower fertility in the Australian rabbit population.[133]

Outside of biology scientists have used a genetically modified virus to construct a lithium-ion battery and other nanostructured materials. It is possible to engineer bacteriophages to express modified proteins on their surface and join them up in specific patterns (a technique called phage display). These structures have potential uses for energy storage and generation, biosensing and tissue regeneration with some new materials currently produced including quantum dots, liquid crystals, nanorings and nanofibres.[134] The battery was made by engineering M13 bacteriaophages so they would coat themselves in iron phosphate and then assemble themselves along a carbon nanotube. This created a highly conductive medium for use in a cathode, allowing energy to be transferred quickly. They could be constructed at lower temperatures with non-toxic chemicals, making them more environmentally friendly.[135]

Fungi can be used for many of the same processes as bacteria. For industrial applications, yeasts combine the bacterial advantages of being a single-celled organism that is easy to manipulate and grow with the advanced protein modifications found in eukaryotes. They can be used to produce large complex molecules for use in food, pharmaceuticals, hormones, and steroids.[136] Yeast is important for wine production and as of 2016 two genetically modified yeasts involved in the fermentation of wine have been commercialized in the United States and Canada. One has increased malolactic fermentation efficiency, while the other prevents the production of dangerous ethyl carbamate compounds during fermentation.[96] There have also been advances in the production of biofuel from genetically modified fungi.[137]

Fungi, being the most common pathogens of insects, make attractive biopesticides. Unlike bacteria and viruses they have the advantage of infecting the insects by contact alone, although they are out competed in efficiency by chemical pesticides. Genetic engineering can improve virulence, usually by adding more virulent proteins,[138] increasing infection rate or enhancing spore persistence.[139] Many of the disease carrying vectors are susceptible to entomopathogenic fungi. An attractive target for biological control are mosquitos, vectors for a range of deadly diseases, including malaria, yellow fever and dengue fever. Mosquitos can evolve quickly so it becomes a balancing act of killing them before the Plasmodium they carry becomes the infectious disease, but not so fast that they become resistant to the fungi. By genetically engineering fungi like Metarhizium anisopliae and Beauveria bassiana to delay the development of mosquito infectiousness the selection pressure to evolve resistance is reduced.[140] Another strategy is to add proteins to the fungi that block transmission of malaria[140] or remove the Plasmodium altogether.[141]

Agaricus bisporus the common white button mushroom, has been gene edited to resist browning, giving it a longer shelf life. The process used CRISPR to knock out a gene that encodes polyphenol oxidase. As it didn't introduce any foreign DNA into the organism it was not deemed to be regulated under existing GMO frameworks and as such is the first CRISPR-edited organism to be approved for release.[142] This has intensified debates as to whether gene-edited organisms should be considered genetically modified organisms[143] and how they should be regulated.[144]

Plants have been engineered for scientific research, to display new flower colors, deliver vaccines, and to create enhanced crops. Many plants are pluripotent, meaning that a single cell from a mature plant can be harvested and under the right conditions can develop into a new plant. This ability can be taken advantage of by genetic engineers; by selecting for cells that have been successfully transformed in an adult plant a new plant can then be grown that contains the transgene in every cell through a process known as tissue culture.[145]

Much of the advances in the field of genetic engineering has come from experimentation with tobacco. Major advances in tissue culture and plant cellular mechanisms for a wide range of plants has originated from systems developed in tobacco.[146] It was the first plant to be altered using genetic engineering and is considered a model organism for not only genetic engineering, but a range of other fields.[147] As such the transgenic tools and procedures are well established making tobacco one of the easiest plants to transform.[148] Another major model organism relevant to genetic engineering is Arabidopsis thaliana. Its small genome and short life cycle makes it easy to manipulate and it contains many homologs to important crop species.[149] It was the first plant sequenced, has a host of online resources available and can be transformed by simply dipping a flower in a transformed Agrobacterium solution.[150]

In research, plants are engineered to help discover the functions of certain genes. The simplest way to do this is to remove the gene and see what phenotype develops compared to the wild type form. Any differences are possibly the result of the missing gene. Unlike mutagenisis, genetic engineering allows targeted removal without disrupting other genes in the organism.[145] Some genes are only expressed in certain tissues, so reporter genes, like GUS, can be attached to the gene of interest allowing visualization of the location.[151] Other ways to test a gene is to alter it slightly and then return it to the plant and see if it still has the same effect on phenotype. Other strategies include attaching the gene to a strong promoter and see what happens when it is overexpressed, forcing a gene to be expressed in a different location or at different developmental stages.[145]

Some genetically modified plants are purely ornamental. They are modified for flower color, fragrance, flower shape and plant architecture.[152] The first genetically modified ornamentals commercialized altered color.[153] Carnations were released in 1997, with the most popular genetically modified organism, a blue rose (actually lavender or mauve) created in 2004.[154] The roses are sold in Japan, the United States, and Canada.[155][156] Other genetically modified ornamentals include Chrysanthemum and Petunia.[152] As well as increasing aesthetic value there are plans to develop ornamentals that use less water or are resistant to the cold, which would allow them to be grown outside their natural environments.[157]

It has been proposed to genetically modify some plant species threatened by extinction to be resistant to invasive plants and diseases, such as the emerald ash borer in North American and the fungal disease, Ceratocystis platani, in European plane trees.[158] The papaya ringspot virus devastated papaya trees in Hawaii in the twentieth century until transgenic papaya plants were given pathogen-derived resistance.[159] However, genetic modification for conservation in plants remains mainly speculative. A unique concern is that a transgenic species may no longer bear enough resemblance to the original species to truly claim that the original species is being conserved. Instead, the transgenic species may be genetically different enough to be considered a new species, thus diminishing the conservation worth of genetic modification.[158]

Genetically modified crops are genetically modified plants that are used in agriculture. The first crops developed were used for animal or human food and provide resistance to certain pests, diseases, environmental conditions, spoilage or chemical treatments (e.g. resistance to a herbicide). The second generation of crops aimed to improve the quality, often by altering the nutrient profile. Third generation genetically modified crops could be used for non-food purposes, including the production of pharmaceutical agents, biofuels, and other industrially useful goods, as well as for bioremediation.[160]

There are three main aims to agricultural advancement; increased production, improved conditions for agricultural workers and sustainability. GM crops contribute by improving harvests through reducing insect pressure, increasing nutrient value and tolerating different abiotic stresses. Despite this potential, as of 2018, the commercialized crops are limited mostly to cash crops like cotton, soybean, maize and canola and the vast majority of the introduced traits provide either herbicide tolerance or insect resistance.[160] Soybeans accounted for half of all genetically modified crops planted in 2014.[161] Adoption by farmers has been rapid, between 1996 and 2013, the total surface area of land cultivated with GM crops increased by a factor of 100.[162] Geographically though the spread has been uneven, with strong growth in the Americas and parts of Asia and little in Europe and Africa.[160] Its socioeconomic spread has been more even, with approximately 54% of worldwide GM crops grown in developing countries in 2013.[162] Although doubts have been raised,[163] most studies have found growing GM crops to be beneficial to farmers through decreased pesticide use as well as increased crop yield and farm profit.[164][165][166]

The majority of GM crops have been modified to be resistant to selected herbicides, usually a glyphosate or glufosinate based one. Genetically modified crops engineered to resist herbicides are now more available than conventionally bred resistant varieties;[167] in the USA 93% of soybeans and most of the GM maize grown is glyphosate tolerant.[168] Most currently available genes used to engineer insect resistance come from the Bacillus thuringiensis bacterium and code for delta endotoxins. A few use the genes that encode for vegetative insecticidal proteins.[169] The only gene commercially used to provide insect protection that does not originate from B. thuringiensis is the Cowpea trypsin inhibitor (CpTI). CpTI was first approved for use cotton in 1999 and is currently undergoing trials in rice.[170][171] Less than one percent of GM crops contained other traits, which include providing virus resistance, delaying senescence and altering the plants composition.[161]

Golden rice is the most well known GM crop that is aimed at increasing nutrient value. It has been engineered with three genes that biosynthesise beta-carotene, a precursor of vitamin A, in the edible parts of rice.[70] It is intended to produce a fortified food to be grown and consumed in areas with a shortage of dietary vitamin A,[172] a deficiency which each year is estimated to kill 670,000 children under the age of 5[173] and cause an additional 500,000 cases of irreversible childhood blindness.[174] The original golden rice produced 1.6g/g of the carotenoids, with further development increasing this 23 times.[175] It gained its first approvals for use as food in 2018.[176]

Plants and plant cells have been genetically engineered for production of biopharmaceuticals in bioreactors, a process known as pharming. Work has been done with duckweed Lemna minor,[177] the algae Chlamydomonas reinhardtii[178] and the moss Physcomitrella patens.[179][180] Biopharmaceuticals produced include cytokines, hormones, antibodies, enzymes and vaccines, most of which are accumulated in the plant seeds. Many drugs also contain natural plant ingredients and the pathways that lead to their production have been genetically altered or transferred to other plant species to produce greater volume.[181] Other options for bioreactors are biopolymers[182] and biofuels.[183] Unlike bacteria, plants can modify the proteins post-translationally, allowing them to make more complex molecules. They also pose less risk of being contaminated.[184] Therapeutics have been cultured in transgenic carrot and tobacco cells,[185] including a drug treatment for Gaucher's disease.[186]

Vaccine production and storage has great potential in transgenic plants. Vaccines are expensive to produce, transport, and administer, so having a system that could produce them locally would allow greater access to poorer and developing areas.[181] As well as purifying vaccines expressed in plants it is also possible to produce edible vaccines in plants. Edible vaccines stimulate the immune system when ingested to protect against certain diseases. Being stored in plants reduces the long-term cost as they can be disseminated without the need for cold storage, don't need to be purified, and have long term stability. Also being housed within plant cells provides some protection from the gut acids upon digestion. However the cost of developing, regulating, and containing transgenic plants is high, leading to most current plant-based vaccine development being applied to veterinary medicine, where the controls are not as strict.[187]

Genetically modified crops have been proposed as one of the ways to reduce farming-related CO2 emissions due to higher yield, reduced use of pesticides, reduced use of tractor fuel and no tillage. According to a 2021 study, in EU alone widespread adoption of GE crops would reduce greenhouse gas emissions by 33 million tons of CO2 equivalent or 7.5% of total farming-related emissions.[188]

The vast majority of genetically modified animals are at the research stage with the number close to entering the market remaining small.[189] As of 2018 only three genetically modified animals have been approved, all in the USA. A goat and a chicken have been engineered to produce medicines and a salmon has increased its own growth.[190] Despite the differences and difficulties in modifying them, the end aims are much the same as for plants. GM animals are created for research purposes, production of industrial or therapeutic products, agricultural uses, or improving their health. There is also a market for creating genetically modified pets.[191]

The process of genetically engineering mammals is slow, tedious, and expensive. However, new technologies are making genetic modifications easier and more precise.[192] The first transgenic mammals were produced by injecting viral DNA into embryos and then implanting the embryos in females.[60] The embryo would develop and it would be hoped that some of the genetic material would be incorporated into the reproductive cells. Then researchers would have to wait until the animal reached breeding age and then offspring would be screened for the presence of the gene in every cell. The development of the CRISPR-Cas9 gene editing system as a cheap and fast way of directly modifying germ cells, effectively halving the amount of time needed to develop genetically modified mammals.[193]

Mammals are the best models for human disease, making genetic engineered ones vital to the discovery and development of cures and treatments for many serious diseases. Knocking out genes responsible for human genetic disorders allows researchers to study the mechanism of the disease and to test possible cures. Genetically modified mice have been the most common mammals used in biomedical research, as they are cheap and easy to manipulate. Pigs are also a good target as they have a similar body size and anatomical features, physiology, pathophysiological response and diet.[194] Nonhuman primates are the most similar model organisms to humans, but there is less public acceptance towards using them as research animals.[195] In 2009, scientists announced that they had successfully transferred a gene into a primate species (marmosets) for the first time.[196][197] Their first research target for these marmosets was Parkinson's disease, but they were also considering amyotrophic lateral sclerosis and Huntington's disease.[198]

Human proteins expressed in mammals are more likely to be similar to their natural counterparts than those expressed in plants or microorganisms. Stable expression has been accomplished in sheep, pigs, rats and other animals. In 2009, the first human biological drug produced from such an animal, a goat, was approved. The drug, ATryn, is an anticoagulant which reduces the probability of blood clots during surgery or childbirth and is extracted from the goat's milk.[199] Human alpha-1-antitrypsin is another protein that has been produced from goats and is used in treating humans with this deficiency.[200] Another medicinal area is in creating pigs with greater capacity for human organ transplants (xenotransplantation). Pigs have been genetically modified so that their organs can no longer carry retroviruses[201] or have modifications to reduce the chance of rejection.[202][203] Pig lungs from genetically modified pigs are being considered for transplantation into humans.[204][205] There is even potential to create chimeric pigs that can carry human organs.[194][206]

Livestock are modified with the intention of improving economically important traits such as growth-rate, quality of meat, milk composition, disease resistance and survival. Animals have been engineered to grow faster, be healthier[207] and resist diseases.[208] Modifications have also improved the wool production of sheep and udder health of cows.[189] Goats have been genetically engineered to produce milk with strong spiderweb-like silk proteins in their milk.[209] A GM pig called Enviropig was created with the capability of digesting plant phosphorus more efficiently than conventional pigs.[210][211] They could reduce water pollution since they excrete 30 to 70% less phosphorus in manure.[210][212] Dairy cows have been genetically engineered to produce milk that would be the same as human breast milk.[213] This could potentially benefit mothers who cannot produce breast milk but want their children to have breast milk rather than formula.[214][215] Researchers have also developed a genetically engineered cow that produces allergy-free milk.[216]

Scientists have genetically engineered several organisms, including some mammals, to include green fluorescent protein (GFP), for research purposes.[217] GFP and other similar reporting genes allow easy visualization and localization of the products of the genetic modification.[218] Fluorescent pigs have been bred to study human organ transplants, regenerating ocular photoreceptor cells, and other topics.[219] In 2011, green-fluorescent cats were created to help find therapies for HIV/AIDS and other diseases[220] as feline immunodeficiency virus is related to HIV.[221]

There have been suggestions that genetic engineering could be used to bring animals back from extinction. It involves changing the genome of a close living relative to resemble the extinct one and is currently being attempted with the passenger pigeon.[222] Genes associated with the woolly mammoth have been added to the genome of an African Elephant, although the lead researcher says he has no intention of creating live elephants and transferring all the genes and reversing years of genetic evolution is a long way from being feasible.[223][224] It is more likely that scientists could use this technology to conserve endangered animals by bringing back lost diversity or transferring evolved genetic advantages from adapted organisms to those that are struggling.[225]

Gene therapy[226] uses genetically modified viruses to deliver genes which can cure disease in humans. Although gene therapy is still relatively new, it has had some successes. It has been used to treat genetic disorders such as severe combined immunodeficiency,[227] and Leber's congenital amaurosis.[228] Treatments are also being developed for a range of other currently incurable diseases, such as cystic fibrosis,[229] sickle cell anemia,[230] Parkinson's disease,[231][232] cancer,[233][234][235] diabetes,[236] heart disease[237] and muscular dystrophy.[238] These treatments only effect somatic cells, meaning any changes would not be inheritable. Germline gene therapy results in any change being inheritable, which has raised concerns within the scientific community.[239][240]

In 2015, CRISPR was used to edit the DNA of non-viable human embryos.[241][242] In November 2018, He Jiankui announced that he had edited the genomes of two human embryos, in an attempt to disable the CCR5 gene, which codes for a receptor that HIV uses to enter cells. He said that twin girls, Lulu and Nana, had been born a few weeks earlier and that they carried functional copies of CCR5 along with disabled CCR5 (mosaicism) and were still vulnerable to HIV. The work was widely condemned as unethical, dangerous, and premature.[243]

Genetically modified fish are used for scientific research, as pets and as a food source. Aquaculture is a growing industry, currently providing over half the consumed fish worldwide.[245] Through genetic engineering it is possible to increase growth rates, reduce food intake, remove allergenic properties, increase cold tolerance and provide disease resistance. Fish can also be used to detect aquatic pollution or function as bioreactors.[246]

Several groups have been developing zebrafish to detect pollution by attaching fluorescent proteins to genes activated by the presence of pollutants. The fish will then glow and can be used as environmental sensors.[247][248] The GloFish is a brand of genetically modified fluorescent zebrafish with bright red, green, and orange fluorescent color. It was originally developed by one of the groups to detect pollution, but is now part of the ornamental fish trade, becoming the first genetically modified animal to become publicly available as a pet when in 2003 it was introduced for sale in the USA.[249]

GM fish are widely used in basic research in genetics and development. Two species of fish, zebrafish and medaka, are most commonly modified because they have optically clear chorions (membranes in the egg), rapidly develop, and the one-cell embryo is easy to see and microinject with transgenic DNA.[250] Zebrafish are model organisms for developmental processes, regeneration, genetics, behavior, disease mechanisms and toxicity testing.[251] Their transparency allows researchers to observe developmental stages, intestinal functions and tumour growth.[252][253] The generation of transgenic protocols (whole organism, cell or tissue specific, tagged with reporter genes) has increased the level of information gained by studying these fish.[254]

GM fish have been developed with promoters driving an over-production of growth hormone for use in the aquaculture industry to increase the speed of development and potentially reduce fishing pressure on wild stocks. This has resulted in dramatic growth enhancement in several species, including salmon,[255] trout[256] and tilapia.[257] AquaBounty Technologies, a biotechnology company, have produced a salmon (called AquAdvantage salmon) that can mature in half the time as wild salmon.[258] It obtained regulatory approval in 2015, the first non-plant GMO food to be commercialized.[259] As of August 2017, GMO salmon is being sold in Canada.[260] Sales in the US started in May 2021.[261]

In biological research, transgenic fruit flies (Drosophila melanogaster) are model organisms used to study the effects of genetic changes on development.[263] Fruit flies are often preferred over other animals due to their short life cycle and low maintenance requirements. They also have a relatively simple genome compared to many vertebrates, with typically only one copy of each gene, making phenotypic analysis easy.[264] Drosophila have been used to study genetics and inheritance, embryonic development, learning, behavior, and aging.[265] The discovery of transposons, in particular the p-element, in Drosophila provided an early method to add transgenes to their genome, although this has been taken over by more modern gene-editing techniques.[266]

Due to their significance to human health, scientists are looking at ways to control mosquitoes through genetic engineering. Malaria-resistant mosquitoes have been developed in the laboratory by inserting a gene that reduces the development of the malaria parasite[267] and then use homing endonucleases to rapidly spread that gene throughout the male population (known as a gene drive).[268][269] This approach has been taken further by using the gene drive to spread a lethal gene.[270][271] In trials the populations of Aedes aegypti mosquitoes, the single most important carrier of dengue fever and Zika virus, were reduced by between 80% and by 90%.[272][273][271] Another approach is to use a sterile insect technique, whereby males genetically engineered to be sterile out compete viable males, to reduce population numbers.[274]

Other insect pests that make attractive targets are moths. Diamondback moths cause US$4 to $5 billion of damage each year worldwide.[275] The approach is similar to the sterile technique tested on mosquitoes, where males are transformed with a gene that prevents any females born from reaching maturity.[276] They underwent field trials in 2017.[275] Genetically modified moths have previously been released in field trials.[277] In this case a strain of pink bollworm that were sterilized with radiation were genetically engineered to express a red fluorescent protein making it easier for researchers to monitor them.[278]

Silkworm, the larvae stage of Bombyx mori, is an economically important insect in sericulture. Scientists are developing strategies to enhance silk quality and quantity. There is also potential to use the silk producing machinery to make other valuable proteins.[279] Proteins currently developed to be expressed by silkworms include; human serum albumin, human collagen -chain, mouse monoclonal antibody and N-glycanase.[280] Silkworms have been created that produce spider silk, a stronger but extremely difficult to harvest silk,[281] and even novel silks.[282]

Systems have been developed to create transgenic organisms in a wide variety of other animals. Chickens have been genetically modified for a variety of purposes. This includes studying embryo development,[283] preventing the transmission of bird flu[284] and providing evolutionary insights using reverse engineering to recreate dinosaur-like phenotypes.[285] A GM chicken that produces the drug Kanuma, an enzyme that treats a rare condition, in its egg passed US regulatory approval in 2015.[286] Genetically modified frogs, in particular Xenopus laevis and Xenopus tropicalis, are used in developmental biology research. GM frogs can also be used as pollution sensors, especially for endocrine disrupting chemicals.[287] There are proposals to use genetic engineering to control cane toads in Australia.[288][289]

The nematode Caenorhabditis elegans is one of the major model organisms for researching molecular biology.[290] RNA interference (RNAi) was discovered in C. elegans[291] and could be induced by simply feeding them bacteria modified to express double stranded RNA.[292] It is also relatively easy to produce stable transgenic nematodes and this along with RNAi are the major tools used in studying their genes.[293] The most common use of transgenic nematodes has been studying gene expression and localization by attaching reporter genes. Transgenes can also be combined with RNAi techniques to rescue phenotypes, study gene function, image cell development in real time or control expression for different tissues or developmental stages.[293] Transgenic nematodes have been used to study viruses,[294] toxicology,[295] diseases,[296][297] and to detect environmental pollutants.[298]

The gene responsible for albinism in sea cucumbers has been found and used to engineer white sea cucumbers, a rare delicacy. The technology also opens the way to investigate the genes responsible for some of the cucumbers more unusual traits, including hibernating in summer, eviscerating their intestines, and dissolving their bodies upon death.[299] Flatworms have the ability to regenerate themselves from a single cell.[300] Until 2017 there was no effective way to transform them, which hampered research. By using microinjection and radiation scientists have now created the first genetically modified flatworms.[301] The bristle worm, a marine annelid, has been modified. It is of interest due to its reproductive cycle being synchronized with lunar phases, regeneration capacity and slow evolution rate.[302] Cnidaria such as Hydra and the sea anemone Nematostella vectensis are attractive model organisms to study the evolution of immunity and certain developmental processes.[303] Other animals that have been genetically modified include snails,[304] geckos, turtles,[305] crayfish, oysters, shrimp, clams, abalone[306] and sponges.[307]

Genetically modified organisms are regulated by government agencies. This applies to research as well as the release of genetically modified organisms, including crops and food. The development of a regulatory framework concerning genetic engineering began in 1975, at Asilomar, California. The Asilomar meeting recommended a set of guidelines regarding the cautious use of recombinant technology and any products resulting from that technology.[308] The Cartagena Protocol on Biosafety was adopted on 29 January 2000 and entered into force on 11 September 2003.[309] It is an international treaty that governs the transfer, handling, and use of genetically modified organisms.[310] One hundred and fifty-seven countries are members of the Protocol and many use it as a reference point for their own regulations.[311]

Universities and research institutes generally have a special committee that is responsible for approving any experiments that involve genetic engineering. Many experiments also need permission from a national regulatory group or legislation. All staff must be trained in the use of GMOs and all laboratories must gain approval from their regulatory agency to work with GMOs.[312] The legislation covering GMOs are often derived from regulations and guidelines in place for the non-GMO version of the organism, although they are more severe.[313] There is a near-universal system for assessing the relative risks associated with GMOs and other agents to laboratory staff and the community. They are assigned to one of four risk categories based on their virulence, the severity of the disease, the mode of transmission, and the availability of preventive measures or treatments. There are four biosafety levels that a laboratory can fall into, ranging from level 1 (which is suitable for working with agents not associated with disease) to level 4 (working with life-threatening agents). Different countries use different nomenclature to describe the levels and can have different requirements for what can be done at each level.[313]

There are differences in the regulation for the release of GMOs between countries, with some of the most marked differences occurring between the US and Europe.[314] Regulation varies in a given country depending on the intended use of the products of the genetic engineering. For example, a crop not intended for food use is generally not reviewed by authorities responsible for food safety.[315] Some nations have banned the release of GMOs or restricted their use, and others permit them with widely differing degrees of regulation.[316][317][318][319] In 2016, thirty eight countries officially ban or prohibit the cultivation of GMOs and nine (Algeria, Bhutan, Kenya, Kyrgyzstan, Madagascar, Peru, Russia, Venezuela and Zimbabwe) ban their importation.[320] Most countries that do not allow GMO cultivation do permit research using GMOs.[321] Despite regulation, illegal releases have sometimes occurred, due to weakness of enforcement.[8]

The European Union (EU) differentiates between approval for cultivation within the EU and approval for import and processing.[322] While only a few GMOs have been approved for cultivation in the EU a number of GMOs have been approved for import and processing.[323] The cultivation of GMOs has triggered a debate about the market for GMOs in Europe.[324] Depending on the coexistence regulations, incentives for cultivation of GM crops differ.[325] The US policy does not focus on the process as much as other countries, looks at verifiable scientific risks and uses the concept of substantial equivalence.[326] Whether gene edited organisms should be regulated the same as genetically modified organism is debated. USA regulations sees them as separate and does not regulate them under the same conditions, while in Europe a GMO is any organism created using genetic engineering techniques.[28]

One of the key issues concerning regulators is whether GM products should be labeled. The European Commission says that mandatory labeling and traceability are needed to allow for informed choice, avoid potential false advertising[327] and facilitate the withdrawal of products if adverse effects on health or the environment are discovered.[328] The American Medical Association[329] and the American Association for the Advancement of Science[330] say that absent scientific evidence of harm even voluntary labeling is misleading and will falsely alarm consumers. Labeling of GMO products in the marketplace is required in 64 countries.[331] Labeling can be mandatory up to a threshold GM content level (which varies between countries) or voluntary. In Canada and the US labeling of GM food is voluntary,[332] while in Europe all food (including processed food) or feed which contains greater than 0.9% of approved GMOs must be labeled.[333] In 2014, sales of products that had been labeled as non-GMO grew 30 percent to $1.1 billion.[334]

There is controversy over GMOs, especially with regard to their release outside laboratory environments. The dispute involves consumers, producers, biotechnology companies, governmental regulators, non-governmental organizations, and scientists. Many of these concerns involve GM crops and whether food produced from them is safe and what impact growing them will have on the environment. These controversies have led to litigation, international trade disputes, and protests, and to restrictive regulation of commercial products in some countries.[335] Most concerns are around the health and environmental effects of GMOs. These include whether they may provoke an allergic reaction, whether the transgenes could transfer to human cells, and whether genes not approved for human consumption could outcross into the food supply.[336]

There is a scientific consensus[337][338][339][340] that currently available food derived from GM crops poses no greater risk to human health than conventional food,[341][342][343][344][345] but that each GM food needs to be tested on a case-by-case basis before introduction.[346][347][348] Nonetheless, members of the public are much less likely than scientists to perceive GM foods as safe.[349][350][351][352] The legal and regulatory status of GM foods varies by country, with some nations banning or restricting them, and others permitting them with widely differing degrees of regulation.[353][354][355][356]

As late as the 1990s gene flow into wild populations was thought to be unlikely and rare, and if it were to occur, easily eradicated. It was thought that this would add no additional environmental costs or risks no effects were expected other than those already caused by pesticide applications.[357] However, in the decades since, several such examples have been observed. Gene flow between GM crops and compatible plants, along with increased use of broad-spectrum herbicides,[358] can increase the risk of herbicide resistant weed populations.[359] Debate over the extent and consequences of gene flow intensified in 2001 when a paper was published showing transgenes had been found in landrace maize in Mexico, the crop's center of diversity.[360][361] Gene flow from GM crops to other organisms has been found to generally be lower than what would occur naturally.[362] In order to address some of these concerns some GMOs have been developed with traits to help control their spread. To prevent the genetically modified salmon inadvertently breeding with wild salmon, all the fish raised for food are females, triploid, 99% are reproductively sterile, and raised in areas where escaped salmon could not survive.[363][364] Bacteria have also been modified to depend on nutrients that cannot be found in nature,[365] and genetic use restriction technology has been developed, though not yet marketed, that causes the second generation of GM plants to be sterile.[366]

Other environmental and agronomic concerns include a decrease in biodiversity, an increase in secondary pests (non-targeted pests) and evolution of resistant insect pests.[367][368][369] In the areas of China and the US with Bt crops the overall biodiversity of insects has increased and the impact of secondary pests has been minimal.[370] Resistance was found to be slow to evolve when best practice strategies were followed.[370] The impact of Bt crops on beneficial non-target organisms became a public issue after a 1999 paper suggested they could be toxic to monarch butterflies. Follow up studies have since shown that the toxicity levels encountered in the field were not high enough to harm the larvae.[371]

Accusations that scientists are "playing God" and other religious issues have been ascribed to the technology from the beginning.[372] With the ability to genetically engineer humans now possible there are ethical concerns over how far this technology should go, or if it should be used at all.[373] Much debate revolves around where the line between treatment and enhancement is and whether the modifications should be inheritable.[374] Other concerns include contamination of the non-genetically modified food supply,[375][376] the rigor of the regulatory process,[377][378] consolidation of control of the food supply in companies that make and sell GMOs,[379] exaggeration of the benefits of genetic modification,[380] or concerns over the use of herbicides with glyphosate.[381] Other issues raised include the patenting of life[382] and the use of intellectual property rights.[383]

There are large differences in consumer acceptance of GMOs, with Europeans more likely to view GM food negatively than North Americans.[384] GMOs arrived on the scene as the public confidence in food safety, attributed to recent food scares such as Bovine spongiform encephalopathy and other scandals involving government regulation of products in Europe, was low.[385] This along with campaigns run by various non-governmental organizations (NGO) have been very successful in blocking or limiting the use of GM crops.[386] NGOs like the Organic Consumers Association, the Union of Concerned Scientists,[387][388][389] Greenpeace and other groups have said that risks have not been adequately identified and managed[390] and that there are unanswered questions regarding the potential long-term impact on human health from food derived from GMOs. They propose mandatory labeling[391][392] or a moratorium on such products.[379][377][393]

The literature about Biodiversity and the GE food/feed consumption has sometimes resulted in an animated debate regarding the suitability of the experimental designs, the choice of the statistical methods, or the public accessibility of data. Such debate, even if positive and part of the natural process of review by the scientific community, has frequently been distorted by the media and often used politically and inappropriately in anti-GE crops campaigns.

And contrast:

The presented articles suggesting the possible harm of GMOs received high public attention. However, despite their claims, they actually weaken the evidence for the harm and lack of substantial equivalency of studied GMOs. We emphasize that with over 1783 published articles on GMOs over the last 10 years it is expected that some of them should have reported undesired differences between GMOs and conventional crops even if no such differences exist in reality.

Despite various concerns, today, the American Association for the Advancement of Science, the World Health Organization, and many independent international science organizations agree that GMOs are just as safe as other foods. Compared with conventional breeding techniques, genetic engineering is far more precise and, in most cases, less likely to create an unexpected outcome.

GM foods currently available on the international market have passed safety assessments and are not likely to present risks for human health. In addition, no effects on human health have been shown as a result of the consumption of such foods by the general population in the countries where they have been approved. Continuous application of safety assessments based on the Codex Alimentarius principles and, where appropriate, adequate post-market monitoring, should form the basis for ensuring the safety of GM foods.

When seeking to optimize the balance between benefits and risks, it is prudent to err on the side of caution and, above all, learn from accumulating knowledge and experience. Any new technology such as genetic modification must be examined for possible benefits and risks to human health and the environment. As with all novel foods, safety assessments in relation to GM foods must be made on a case-by-case basis.

Members of the GM jury project were briefed on various aspects of genetic modification by a diverse group of acknowledged experts in the relevant subjects. The GM jury reached the conclusion that the sale of GM foods currently available should be halted and the moratorium on commercial growth of GM crops should be continued. These conclusions were based on the precautionary principle and lack of evidence of any benefit. The Jury expressed concern over the impact of GM crops on farming, the environment, food safety and other potential health effects.

The Royal Society review (2002) concluded that the risks to human health associated with the use of specific viral DNA sequences in GM plants are negligible, and while calling for caution in the introduction of potential allergens into food crops, stressed the absence of evidence that commercially available GM foods cause clinical allergic manifestations. The BMA shares the view that there is no robust evidence to prove that GM foods are unsafe but we endorse the call for further research and surveillance to provide convincing evidence of safety and benefit.

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Genetic engineering – DNA Modification, Cloning, Gene Splicing

Posted: November 16, 2023 at 2:51 pm

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

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

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

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

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

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

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

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

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

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18 Human Genetic Engineering – Clemson University

Posted: April 7, 2023 at 12:08 am

Melissa Nolan

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

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

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

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

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

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

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

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

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

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

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

DNA CRISPR Scissors by Max Pixel is licensed under CC0 1.0

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

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

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

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

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

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

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

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

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

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

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

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

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

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

A. Recombinant DNA technology

B. CRISPR/Cas9

C. Bacterial Transformation

D. Immunoglobulin

2. When was gene therapy concepts first introduced?

A. 1920s

B. 1940s

C. 1960s

D. 1980s

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

A. Potential for ageism

B. Gene editing is only 50% effective

C. HGE can only be used in Caucasians

D. Potential for eugenics

Answers:

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

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

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

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

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

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

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

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

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

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18 Human Genetic Engineering - Clemson University

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Pros and Cons of Genetic Engineering – Benefits and Risks

Posted: April 7, 2023 at 12:08 am

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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Pros and Cons of Genetic Engineering - Benefits and Risks

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Genetic Engineering – Meaning, Applications, Advantages and Challenges …

Posted: March 12, 2023 at 12:10 am

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

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

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

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

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

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

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

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

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

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Genetic Engineering Principles of Biology

Posted: December 27, 2022 at 1:13 am

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

OpenStax, Biology. OpenStax CNX. May 27, 2016 http://cnx.org/contents/s8Hh0oOc@9.10:8CA_YwJq@3/Cloning-and-Genetic-Engineerin

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

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Genetic Engineering Principles of Biology

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Engineering the Perfect Baby | MIT Technology Review

Posted: December 27, 2022 at 1:13 am

Indeed, some people are adamant that germ-line engineering is being pushed ahead with false arguments. That is the view of Edward Lanphier, CEO of Sangamo Biosciences, a California biotechnology company that is using another gene-editing technique, called zinc fingers nucleases, to try to treat HIV in adults by altering their blood cells. Weve looked at [germ-line engineering] for a disease rationale, and there is none, he says. You can do it. But there really isnt a medical reason. People say, well, we dont want children born with this, or born with thatbut its a completely false argument and a slippery slope toward much more unacceptable uses.

Critics cite a host of fears. Children would be the subject of experiments. Parents would be influenced by genetic advertising from IVF clinics. Germ-line engineering would encourage the spread of allegedly superior traits. And it would affect people not yet born, without their being able to agree to it. The American Medical Association, for instance, holds that germ-line engineering shouldnt be done at this time because it affects the welfare of future generations and could cause unpredictable and irreversible results. But like a lot of official statements that forbid changing the genome, the AMAs, which was last updated in 1996, predates todays technology. A lot of people just agreed to these statements, says Greely. It wasnt hard to renounce something that you couldnt do.

The fear? A dystopia of superpeople and designer babies for those who can afford it.

Others predict that hard-to-oppose medical uses will be identified. A couple with several genetic diseases at once might not be able to find a suitable embryo. Treating infertility is another possibility. Some men dont produce any sperm, a condition called azoospermia. One cause is a genetic defect in which a region of about one million to six million DNA letters is missing from the Y chromosome. It might be possible to take a skin cell from such a man, turn it into a stem cell, repair the DNA, and then make sperm, says Werner Neuhausser, a young Austrian doctor who splits his time between the Boston IVF fertility-clinic network and Harvards Stem Cell Institute. That will change medicine forever, right? You could cure infertility, that is for sure, he says.

I spoke with Church several times by telephone over the last few months, and he told me whats driving everything is the incredible specificity of CRISPR. Although not all the details have been worked out, he thinks the technology could replace DNA letters essentially without side effects. He says this is what makes it tempting to use. Church says his laboratory is focused mostly on experiments in engineering animals. He added that his lab would not make or edit human embryos, calling such a step not our style.

What is Churchs style is human enhancement. And hes been making a broad case that CRISPR can do more than eliminate disease genes. It can lead to augmentation. At meetings, some involving groups of transhumanists interested in next steps for human evolution, Church likes to show a slide on which he lists naturally occurring variants of around 10 genes that, when people are born with them, confer extraordinary qualities or resistance to disease. One makes your bones so hard theyll break a surgical drill. Another drastically cuts the risk of heart attacks. And a variant of the gene for the amyloid precursor protein, or APP, was found by Icelandic researchers to protect against Alzheimers. People with it never get dementia and remain sharp into old age.

Church thinks CRISPR could be used to provide people with favorable versions of genes, making DNA edits that would act as vaccines against some of the most common diseases we face today. Although he told me anything edgy should be done only to adults who can consent, its obvious to him that the earlier such interventions occur, the better.

Church tends to dodge questions about genetically modified babies. The idea of improving the human species has always had enormously bad press, he wrote in the introduction to Regenesis, his 2012 book on synthetic biology, whose cover was a painting by Eustache Le Sueur of a bearded God creating the world. But thats ultimately what hes suggesting: enhancements in the form of protective genes. An argument will be made that the ultimate prevention is that the earlier you go, the better the prevention, he told an audience at MITs Media Lab last spring. I do think its the ultimate preventive, if we get to the point where its very inexpensive, extremely safe, and very predictable. Church, who has a less cautious side, proceeded to tell the audience that he thought changing genes is going to get to the point where its like you are doing the equivalent of cosmetic surgery.

Some thinkers have concluded that we should not pass up the chance to make improvements to our species. The human genome is not perfect, says John Harris, a bioethicist at Manchester University, in the U.K. Its ethically imperative to positively support this technology. By some measures, U.S. public opinion is not particularly negative toward the idea. A Pew Research survey carried out last August found that 46 percent of adults approved of genetic modification of babies to reduce the risk of serious diseases.

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