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67-422: The discovery of CED-12 was done using knockout experiments . Its involvement in the apoptotic phagocytosis pathway was first noted when knocked-out ced - 12 in C. elegans showed similar results in the apoptotic process to ced-5 and ced-2 knockouts. This lead researchers to believe, and later confirm, that the protein products of ced-12 (CED-12), ced-5 (CED-5), and ced-2 (CED-2) all functioned as part of

134-500: A transgene at a non-specific location in the organisms' genome, as well as gene-editing making small edits to the DNA already present in the organisms, verses genetic modification insertion 'foreign' DNA from another species. Because gene editing makes smaller changes to endogenous DNA, many mutations created through genome-editing could in theory occur through natural mutagenesis or, in the context of plants, through mutation breeding which

201-450: A DNA binding domain and a nuclease that can cleave DNA. The DNA binding region consists of amino acid repeats that each recognize a single base pair of the desired targeted DNA sequence. If this cleavage is targeted to a gene coding region, and NHEJ-mediated repair introduces insertions and deletions, a frameshift mutation often results, thus disrupting function of the gene. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)

268-439: A DNA sequence. Each zinc-finger can recognize codons of a desired DNA sequence, and therefore can be modularly assembled to bind to a particular sequence. These binding domains are coupled with a restriction endonuclease that can cause a double stranded break (DSB) in the DNA. Repair processes may introduce mutations that destroy functionality of the gene. Transcription activator-like effector nucleases ( TALENs ) also contain

335-463: A double stranded break in the DNA. Following the same principle as zinc-fingers and TALENs, the attempts to repair these double stranded breaks often result in frameshift mutations that result in an nonfunctional gene. Non invasive CRISPR-Cas9 technology has successfully knocked out a gene associated in depression and anxiety in mice, being the first successful delivery passing through the blood–brain barrier to enable gene modification. Gene knock-in

402-416: A gene-targeted organism, DNA must be introduced into its cells. This DNA must contain all of the parts necessary to complete the gene targeting. At a minimum this is the homology repair template, containing the desired edit flanked by regions of DNA homologous (identical in sequence to) the targeted region (these homologous regions are called “homology arms” ). Often a reporter gene and/or a selectable marker

469-409: A molecular scissors) to the target cell, and then allowing the cell to repair the cut in the DNA. When the cell repairs the cut, it can either join the cut ends back together, resulting in a non-functional gene, or introduce a mutation that disrupts the gene's function. This technique can be used in a variety of organisms, including bacteria, yeast, plants, and animals, and it allows scientists to study

536-540: A new wave of isogenic human disease models . These models are the most accurate in vitro models available to researchers and facilitate the development of personalized drugs and diagnostics, particularly in oncology . Gene targeting has also been investigated for gene therapy to correct disease-causing mutations. However the low efficiency of delivery of the gene-targeting machinery into cells has hindered this, with research conducted into viral vectors for gene targeting to try and address these challenges. Gene targeting

603-514: A specific gene. Cassettes can be used for many different things while the flanking homology regions of gene targeting cassettes need to be adapted for each gene. This makes gene trapping more easily amenable for large scale projects than targeting. On the other hand, gene targeting can be used for genes with low transcriptions that would go undetected in a trap screen. The probability of trapping increases with intron size, while for gene targeting, small genes are just as easily altered. Gene targeting

670-481: A variety of techniques. Originally, naturally occurring mutations were identified and then gene loss or inactivation had to be established by DNA sequencing or other methods. Gene knockout by mutation is commonly carried out in bacteria. An early instance of the use of this technique in Escherichia coli was published in 1989 by Hamilton, et al. In this experiment, two sequential recombinations were used to delete

737-404: A widely used genetic engineering technique that involves the targeted removal or inactivation of a specific gene within an organism's genome. This can be done through a variety of methods, including homologous recombination , CRISPR-Cas9 , and TALENs . One of the main advantages of gene knockouts is that they allow researchers to study the function of a specific gene in vivo, and to understand

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804-401: Is a genetic engineering technique that allows for precise editing of the genome. One application of CRISPR is gene knockout, which involves disabling or "knocking out" a specific gene in an organism. The process of gene knockout with CRISPR involves three main steps: designing a guide RNA (gRNA) that targets a specific location in the genome, delivering the gRNA and a Cas9 enzyme (which acts as

871-548: Is also required, to help identify and select for cells (or “events”) where GT has actually occurred. It is also common practice to increase GT rates by causing a double-strand-break (DSB) in the targeted DNA region. Hence the genes encoding for the site-specific-nuclease of interest may also be transformed along with the repair template. These genetic elements required for GT may be assembled through conventional molecular cloning in bacteria. Gene targeting methods are established for several model organisms and may vary depending on

938-458: Is an adaptor protein (proteins involved in facilitating the formation of signalling complexes) that is translated once apoptosis has been triggered in a cell. Apoptosis, also known as programmed cell death, activates during development as well as in situations where a cell has received sufficient physical damage. Many of the contents within a cell are reactive with the environment outside of the cell and must be disposed of without causing any harm to

1005-499: Is an error-prone DNA repair pathway, meaning that when it repairs the broken DNA it can insert or delete DNA bases, creating insertions or deletions (indels). The user cannot specify what these random indels will be, hence they cannot control exactly what edits are made at the target site. However they can control where these edits will occur (i.e. dictate the target site) through using a site-specific nuclease (previously Zinc Finger Nucleases & TALENs , now commonly CRISPR ) to break

1072-613: Is an inefficient process, as homologous recombination accounts for only 10 to 10 of DNA integrations. Often, the drug selection marker on the construct is used to select for cells in which the recombination event has occurred. These stem cells now lacking the gene could be used in vivo , for instance in mice, by inserting them into early embryos. If the resulting chimeric mouse contained the genetic change in their germline, this could then be passed on offspring. In diploid organisms, which contain two alleles for most genes, and may as well contain several related genes that collaborate in

1139-415: Is characterised by making small edits to the genome at a specific location, often following cutting of the target DNA region by a site-specific-nuclease such as CRISPR. Genetic modification usually describes the insertion of a transgene (foreign DNA, i.e. a gene from another species) into a random location within the genome. Gene-targeting is a specific biotechnological tool that can lead to small changes to

1206-464: Is distinct from natural homology-directed repair, during which the ‘natural’ DNA repair template of the sister chromatid is used to repair broken DNA (the sister chromatid is the second copy of the gene). The alteration of DNA sequence in an organism can be useful in both a research context – for example to understand the biological role of a gene – and in biotechnology, for example to alter the traits of an organism (e.g. to improve crop plants). To create

1273-548: Is expressed. Both the CED-2/CED-5/CED-12 ternary structure and CED-6 function to activate an effector protein known as CED-10. CED-10 is a RAC-GTPase protein that is directly responsible for the rearrangement of the actin cytoskeleton that initiates phagocytosis. This process is regulated by two pathways. The first is by CED-6, which is an adaptor protein that is responsible for coordinating protein-protein interactions between CED-10 and actin . The second pathway occurs when

1340-410: Is known as a double knockout ( DKO ). Similarly the terms triple knockout ( TKO ) and quadruple knockouts ( QKO ) are used to describe three or four knocked out genes, respectively. However, one needs to distinguish between heterozygous and homozygous KOs. In the former, only one of two gene copies ( alleles ) is knocked out, in the latter both are knocked out. Knockouts are accomplished through

1407-407: Is limited in the length of DNA sequence insertion possible; base editing is limited to single base pair conversions while prime editing can only insert sequences of up to ~44bp. Hence GT remains the primary method of targeted (location-specific) insertion of long DNA sequences for genome engineering.   Gene trapping is based on random insertion of a cassette, while gene targeting manipulates

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1474-524: Is more commonly used to insert smaller sequences. The range of edits possible through GT can make it challenging to regulate (see Regulation ). The two most established forms of gene editing are gene-targeting and targeted-mutagenesis . While gene targeting relies on the Homology Directed Repair (HDR) (also called Homologous Recombination , HR) DNA repair pathway, targeted-mutagenesis uses Non-Homologous-End-Joining (NHEJ) of broken DNA. NHEJ

1541-416: Is one specific form of genome editing tool. Other genome editing tools include targeted mutagenesis, base editing and prime editing , all of which create edits to the endogenous DNA (DNA already present in the organism) at a specific genomic location. This site-specific or ‘targeted’ nature of genome editing is typically what makes genome-editing different to traditional ‘genetic modification’ which inserts

1608-507: Is part of conventional breeding (in contrast the insertion of a transgene to create a Genetically Modified Organism (GMO) could not occur naturally). However, there are exceptions to this general rule; as explained in the introduction, GT can introduce a range of possible size of edits to DNA; from very small edits such as changing, inserting or deleting 1 base-pair, through to inserting much longer DNA sequences, which could in theory include insertion of an entire transgene. However, in practice GT

1675-516: Is particularly challenging in higher plants due to the low rates of Homologous Recombination, or Homology Directed Repair, in higher plants and the low rate of transformation (DNA uptake) by many plant species. However, there has been much effort to increase the frequencies of gene targeting in plants in the past decades, as it is very useful to be able to introduce specific sequences in the plant genome for plant genome engineering. The most significant improvement to gene targeting frequencies in plants

1742-430: Is relatively high efficiency in yeast, bacterial and moss (but is rare in higher eukaryotes). Hence gene targeting has been used in reverse genetics approaches to study gene function in these systems. Gene targeting (GT), or homology-directed repair (HDR), is used routinely in plant genome engineering to insert specific sequences, with the first published example of GT in plants in the 1980s. However, gene targeting

1809-409: Is similar to gene knockout, but it replaces a gene with another instead of deleting it. A conditional gene knockout allows gene deletion in a tissue in a tissue specific manner. This is required in place of a gene knockout if the null mutation would lead to embryonic death , or a specific tissue or cell type is of specific interest. This is done by introducing short sequences called loxP sites around

1876-555: Is technically capable of creating a range of sizes of genetic changes; from single base-pair mutations through to insertion of longer sequences, including potentially transgenes. This means that products of gene targeting can be indistinguishable from natural mutation, or can be equivalent to GMOs due to their insertion of a transgene (see Venn diagram above). Hence regulating products of Gene Targeting can be challenging and different countries have taken different approaches or are reviewing how to do so as part of broader regulatory reviews into

1943-450: Is the exchange of genes between two DNA strands that include extensive regions of base sequences that are identical to one another. In eukaryotic species, bacteria, and some viruses, homologous recombination happens spontaneously and is a useful tool in genetic engineering. Homologous recombination, which takes place during meiosis in eukaryotes, is essential for the repair of double-stranded DNA breaks and promotes genetic variation by allowing

2010-579: The European Union (EU) has broadly been opposed to Genetic Modification technology, on grounds of its precautionary principle . In 2018 the European Court of Justice (ECJ) ruled that gene-edited crops (including gene-targeted crops) should be considered as genetically modified and therefore were subject to the GMO Directive, which places significant regulatory burdens on GMO use. However this decision

2077-410: The species used. To target genes in mice , the DNA is inserted into mouse embryonic stem cells in culture. Cells with the insertion can contribute to a mouse's tissue via embryo injection. Finally, chimeric mice where the modified cells make up the reproductive organs are bred . After this step the entire body of the mouse is based on the selected embryonic stem cell. To target genes in moss ,

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2144-470: The 2007 Nobel Prize in Physiology or Medicine for their findings. Traditionally, homologous recombination was the main method for causing a gene knockout. This method involves creating a DNA construct containing the desired mutation. For knockout purposes, this typically involves a drug resistance marker in place of the desired knockout gene. The construct will also contain a minimum of 2kb of homology to

2211-529: The CED-2/CED-5/CED-12 ternary structure form a GEF (guanine nucleotide exchange factor) with CED-10, which promotes the binding of a GTP energy molecule in order to activate the GTP-dependent CED-10. CED-12 also functions in cell migration processes, which is regulated by the same interactions as the apoptotic phagocytosis pathway. It functions in distal tip cell migration in gonad development in C. elegans. Distal tip cells are somatic cells located at

2278-701: The Chinese Han Population. For gene knockout investigations, RNA interference (RNAi), a more recent method, also known as gene silencing, has gained popularity. In RNA interference (RNAi), messenger RNA for a particular gene is inactivated using small interfering RNA (siRNA) or short hairpin RNA (shRNA). This effectively stops the gene from being expressed. Oncogenes like Bcl-2 and p53, as well as genes linked to neurological disease, genetic disorders, and viral infections, have all been targeted for gene silencing utilizing RNA interference (RNAi). Homologous recombination

2345-418: The DNA at the target site. A summary of gene-targeting through HDR (also called Homologous Recombination) and targeted mutagenesis through NHEJ is shown in the figure below. The more newly developed gene-editing techniques of prime editing and base editing, based on CRISPR-Cas methods, are alternatives to gene targeting, which can also create user-defined edits at targeted genomic locations. However each

2412-509: The DNA is incubated together with freshly isolated protoplasts and with polyethylene glycol . As mosses are haploid organisms, moss filaments ( protonema ) can be directly screened for the target, either by treatment with antibiotics or with PCR . Unique among plants , this procedure for reverse genetics is as efficient as in yeast . Gene targeting has been successfully applied to cattle, sheep, swine and many fungi. The frequency of gene targeting can be significantly enhanced through

2479-634: The cell detects the similar flanking regions as homologues. The target gene is "knocked out" by the exchange. By using this technique to target particular alleles in embryonic stem cells in mice, it is possible to create knockout mice. With the aid of gene targeting, numerous mouse genes have been shut down, leading to the creation of hundreds of distinct mouse models of various human diseases, such as cancer, diabetes, cardiovascular diseases, and neurological disorders. Mario Capecchi, Sir Martin J. Evans, and Oliver Smithies performed groundbreaking research on homologous recombination in mouse stem cells, and they shared

2546-401: The cell membrane. CED-12 binds CED-2 ( C. elegans homolog to CrkII in mammals), followed by CED-5 ( C. elegans homolog for DOCK180 in mammals) and forms a ternary structure . Transmembrane CED-1 is an example of the cell-surface receptor on the engulfing cell. When receptors come in contact with cell surface markers on the apoptotic cell, a protein known as CED-6 (homolog for GULP in mammals)

2613-530: The distal tip cells. The ced-12 gene codes for an 82kDa large protein, which spans 731 amino acids in length. It is found on chromosome 2 on the L-arm in Drosophila , and on chromosome I in C. elegans . The protein structure of CED-12 is separated based on its binding domains: CED-12 has been shown to interact with: Gene knockout Gene knockouts (also known as gene deletion or gene inactivation ) are

2680-623: The entire genome , such as in Saccharomyces cerevisiae . Gene targeting Gene targeting is a biotechnological tool used to change the DNA sequence of an organism (hence it is a form of Genome Editing ). It is based on the natural DNA-repair mechanism of Homology Directed Repair (HDR), including Homologous Recombination . Gene targeting can be used to make a range of sizes of DNA edits, from larger DNA edits such as inserting entire new genes into an organism, through to much smaller changes to

2747-439: The existing DNA such as a single base-pair change. Gene targeting relies on the presence of a repair template to introduce the user-defined edits to the DNA. The user (usually a scientist) will design the repair template to contain the desired edit, flanked by DNA sequence corresponding (homologous) to the region of DNA that the user wants to edit; hence the edit is targeted to a particular genomic region. In this way Gene Targeting

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2814-441: The function of specific genes by observing the effects of their absence. CRISPR-based gene knockout is a powerful tool for understanding the genetic basis of disease and for developing new therapies. It is important to note that CRISPR-based gene knockout, like any genetic engineering technique, has the potential to produce unintended or harmful effects on the organism, so it should be used with caution. The coupled Cas9 will cause

2881-418: The function of specific genes in development, physiology, and cancer research. The use of gene knockouts in mouse models has been particularly valuable in the study of human diseases. For example, gene knockouts in mice have been used to study the role of specific genes in cancer, neurological disorders, immune disorders, and metabolic disorders. However, gene knockouts also have some limitations. For example,

2948-413: The gene to be turned off and on at specific times or in specific tissues. Conditional knockouts are particularly useful for studying developmental processes and for understanding the role of a gene in specific cell types or tissues. Gene knockouts have been widely used in many different organisms, including bacteria, yeast, fruit flies, zebrafish, and mice. In mice, gene knockouts are commonly used to study

3015-446: The gene. These sequences will be introduced into the germ-line via the same mechanism as a knockout. This germ-line can then be crossed to another germline containing Cre-recombinase which is a viral enzyme that can recognize these sequences, recombines them and deletes the gene flanked by these sites. Other recombinases have since been created and employed in conditional knockout experiments. Knockouts are primarily used to understand

3082-572: The gene. This work established the feasibility of removing or replacing a functional gene in bacteria. That method has since been developed for other organisms, particularly research animals, like mice. Knockout mice are commonly used to study genes with human equivalents that may have significance for disease. An example of a study using knockout mice is an investigation of the roles of Xirp proteins in Sudden Unexplained Nocturnal Death Syndrome (SUNDS) and Brugada Syndrome in

3149-513: The genome at a specific site - in which case the edits caused by gene-targeting would count as genome editing. However gene targeting is also capable of inserting entire genes (such as transgenes) at the target site if the transgene is incorporated into the homology repair template that is used during gene-targeting. In such cases the edits caused by gene-targeting would, in some jurisdictions, be considered as equivalent to Genetic Modification as insertion of foreign DNA has occurred. Gene targeting

3216-449: The genome. However its primary applications - human disease modelling and plant genome engineering - are hindered by the low efficiency of homologous recombination in comparison to the competing non-homologous end joining in mammalian and higher plant cells. As described above, there are strategies that can be employed to increase the frequencies of gene targeting in plants and mammalian cells. In addition, robust selection methods that allow

3283-473: The homologous repair template; and engineering Cas variants to be optimised for plant tissue culture. Some of these approaches have also been used to improve gene targeting efficiencies in mammalian cells. Plants that have been gene-targeted include Arabidopsis thaliana (the most commonly used model plant ), rice, tomato, maize, tobacco and wheat. Gene targeting holds enormous promise to make targeted, user-defined sequence changes or sequence insertions in

3350-409: The introduction of an engineered mutation into a particular gene in order to learn more about the function of that gene. This method involves inserting foreign DNA into a cell that has a sequence similar to the target gene while being flanked by sequences that are the same upstream and downstream of the target gene. The target gene's DNA is substituted with the foreign DNA sequence during replication when

3417-466: The loss of a single gene may not fully mimic the effects of a genetic disorder, and the knockouts may have unintended effects on other genes or pathways. Additionally, gene knockouts are not always a good model for human disease as the mouse genome is not identical to the human genome, and mouse physiology is different from human physiology. The KO technique is essentially the opposite of a gene knock-in . Knocking out two genes simultaneously in an organism

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3484-414: The movement of genetic information during chromosomal crossing. Homologous recombination, a key DNA repair mechanism in bacteria, enables the insertion of genetic material acquired through horizontal transfer of genes and transformation into DNA. Homologous recombination in viruses influences the course of viral evolution. Homologous recombination, a type of gene targeting used in genetic engineering, involves

3551-431: The products of gene-editing. Broadly adopted classifications split gene-edited organisms into 3 classes of "SDN1-3", referring to Site Directed Nucleases (such as CRISPR-Cas) that are used to generate gene-edited organisms. These SDN classifications can guide national regulations as to which class of SDN they will consider to be ‘GMOs’ and therefore which are subject to potentially strict regulations.  Historically

3618-424: The role of a specific gene or DNA region by comparing the knockout organism to a wildtype with a similar genetic background. Knockout organisms are also used as screening tools in the development of drugs , to target specific biological processes or deficiencies by using a specific knockout, or to understand the mechanism of action of a drug by using a library of knockout organisms spanning

3685-435: The role of the gene in normal development and physiology as well as in the pathology of diseases. By studying the phenotype of the organism with the knocked out gene, researchers can gain insights into the biological processes that the gene is involved in. There are two main types of gene knockouts: complete and conditional. A complete gene knockout permanently inactivates the gene, while a conditional gene knockout allows for

3752-419: The same pathway. Researchers also noted direct protein-protein interactions between CED-12 and CED-10 ( C. elegans homolog for Rac1 ), a Rac-GTPase (energy-dependent protein found used for cytoskeletal rearrangements among other functions). CED-10 was inactive when CED-12 was knocked-out. Expression of CED-12 with CED-5 and CED-2 activated CED-10, which lead to the activation of apoptotic phagocytosis. CED-12

3819-519: The same role, additional rounds of transformation and selection are performed until every targeted gene is knocked out. Selective breeding may be required to produce homozygous knockout animals. There are currently three methods in use that involve precisely targeting a DNA sequence in order to introduce a double-stranded break. Once this occurs, the cell's repair mechanisms will attempt to repair this double stranded break, often through non-homologous end joining (NHEJ), which involves directly ligating

3886-440: The selection or specific enrichment of cells where gene targeting has occurred can increase the rates of recovery of gene-targeted cells. Mario R. Capecchi , Martin J. Evans and Oliver Smithies were awarded the 2007 Nobel Prize in Physiology or Medicine for their work on "principles for introducing specific gene modifications in mice by the use of embryonic stem cells", or gene targeting. As explained above, Gene Targeting

3953-510: The surface of the distal tip cells meet chemoattractants located on the extracellular matrix . The integrins form focal adhesions at the sites of the chemoattractants, which causes the localization of CED-5 to the adhesion points. CED-12 and CED-2 form the GEF-trio with CED-5 and activate the CED-10 Rac-GTPase in order to rearrange the actin cytoskeleton and promote the forward propagation of

4020-488: The surrounding tissues. Apoptotic cells are removed from their external environment by neighbouring cells that recognize cell-surface markers located on the apoptotic cell membrane. Marker recognition leads to the engulfment of apoptotic cells by phagocytosis. On a molecular level, recognition of the cell-surface markers leads to the translation of the CED-12 protein in the cytoplasm of the engulfing cell, which then gets localized to

4087-410: The target sequence. The construct can be delivered to stem cells either through microinjection or electroporation . This method then relies on the cell's own repair mechanisms to recombine the DNA construct into the existing DNA. This results in the sequence of the gene being altered, and most cases the gene will be translated into a nonfunctional protein , if it is translated at all. However, this

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4154-403: The tip of developing gonadal arms, and are responsible for the elongation of the gonadal arm as well as controlling mitotic and meiotic cell division of gonadal cells throughout development and adulthood. As C. elegans develops, the distal cells undergo a series of migrations in order to complete morphological changes, which define both gonad shape and size. This process occurs when integrins on

4221-483: The two cut ends together. This may be done imperfectly, therefore sometimes causing insertions or deletions of base pairs, which cause frameshift mutations . These mutations can render the gene in which they occur nonfunctional, thus creating a knockout of that gene. This process is more efficient than homologous recombination, and therefore can be more easily used to create biallelic knockouts. Zinc-finger nucleases consist of DNA binding domains that can precisely target

4288-607: The use of site-specific endonucleases such as zinc finger nucleases , engineered homing endonucleases , TALENS , or most commonly the CRISPR -Cas system. This method has been applied to species including Drosophila melanogaster , tobacco , corn , human cells, mice and rats . The relationship between gene targeting, gene editing and genetic modification is outlined in the Venn diagram below. It displays how 'Genetic engineering' encompasses all 3 of these techniques. Genome editing

4355-518: Was developed in mammalian cells in the 1980s, with diverse applications possible as a result of being able to make specific sequence changes at a target genomic site, such as the study of gene function or human disease, particularly in mice models. Indeed, gene targeting has been widely used to study human genetic diseases by removing (" knocking out "), or adding (" knocking in "), specific mutations of interest. Previously used to engineer rat cell models, advances in gene targeting technologies enable

4422-617: Was received negatively by the European scientific community. In 2021 the European Commission deemed that current EU legislation governing Genetic Modification and Gene-Editing techniques (or NGTs – New Genomic Techniques) was ‘not fit for purpose’ and needed adapting to reflect scientific and technological progress. In July 2023 the European Commission published a proposal to change rules for certain products of gene-editing to reduce

4489-445: Was the induction of double-strand-breaks through site specific nucleases such as CRISPR, as described above. Other strategies include in planta gene targeting, whereby the homology repair template is embedded within the plant genome and then liberated using CRISPR cutting; upregulation of genes involved in the homologous recombination pathway; downregulation of the competing Non-Homologous-End-Joining pathway; increasing copy numbers of

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