genome editing


  • [3] It has also enabled the editing of specific sequences within a genome as well as reduced off target effects.

  • [22] Zinc fingers have been more established in these terms and approaches such as modular assembly (where Zinc fingers correlated with a triplet sequence are attached in
    a row to cover the required sequence), OPEN (low-stringency selection of peptide domains vs. triplet nucleotides followed by high-stringency selections of peptide combination vs. the final target in bacterial systems), and bacterial one-hybrid
    screening of zinc finger libraries among other methods have been used to make site specific nucleases.

  • [31] It is also possible to fuse a protein constructed in this way with the catalytic domain of an endonuclease in order to induce a targeted DNA break, and therefore to use
    these proteins as genome engineering tools.

  • By crossing an organism containing the recombinase sites flanking the gene of interest with an organism that express the SSR under control of tissue specific promoters, it
    is possible to knock out or switch on genes only in certain cells.

  • [17] By creating DNA constructs that contain a template that matches the targeted genome sequence it is possible that the HR processes within the cell will insert the construct
    at the desired location.

  • [37] TALEN constructs are used in a similar way to designed zinc finger nucleases, and have three advantages in targeted mutagenesis: (1) DNA binding specificity is higher,
    (2) off-target effects are lower, and (3) construction of DNA-binding domains is easier.

  • Although, several methods have been discovered which target the inserted genes to specific sites within an organism genome.

  • [29] Meganucleases have the benefit of causing less toxicity in cells than methods such as Zinc finger nuclease (ZFN), likely because of more stringent DNA sequence recognition;[23]
    however, the construction of sequence-specific enzymes for all possible sequences is costly and time-consuming, as one is not benefiting from combinatorial possibilities that methods such as ZFNs and TALEN-based fusions utilize.

  • Meganucleases, found commonly in microbial species, have the unique property of having very long recognition sequences (>14bp) thus making them naturally very specific.

  • One drawback of this technology has been the random nature with which the DNA is inserted into the hosts genome, which can impair or alter other genes within the organism.

  • In order to study the function of these genes site specific recombinases were used.

  • Although the direct genome-wide characterization of zinc finger nuclease activity has not been reported, an assay that measures the total number of double-strand DNA breaks
    in cells found that only one to two such breaks occur above background in cells treated with zinc finger nucleases with a 24 bp composite recognition site and obligate heterodimer FokI nuclease domains.

  • [27] Another approach involves using computer models to try to predict as accurately as possible the activity of the modified meganucleases and the specificity of the recognized
    nucleic sequence.

  • Various selection techniques, using bacteria, yeast or mammal cells have been developed to identify the combinations that offer the best specificity and the best cell tolerance.

  • Further modifications of these systems allowed researchers to induce recombination only under certain conditions, allowing genes to be knocked out or expressed at desired
    times or stages of development.

  • [25][26] Yet others have attempted to alter the DNA interacting aminoacids of the meganuclease to design sequence specific meganucelases in a method named rationally designed

  • This could be used for research purposes, by targeting mutations to specific genes, and in gene therapy.

  • By inserting a functional gene into an organism and targeting it to replace the defective one it could be possible to cure certain genetic diseases.

  • Commonly used restriction enzymes are effective at cutting DNA, but generally recognize and cut at multiple sites.

  • Matching colors signify DNA recognition patterns The key to genome editing is creating a DSB at a specific point within the genome.

  • all three major classes of these enzymes—zinc finger nucleases, transcription activator-like effector nucleases and engineered meganucleases—were selected by Nature Methods
    as the 2011 Method of the Year.

  • Zinc finger nucleases[edit] As opposed to meganucleases, the concept behind ZFNs and TALEN technology is based on a non-specific DNA cutting catalytic domain, which can then
    be linked to specific DNA sequence recognizing peptides such as zinc fingers and transcription activator-like effectors (TALEs).

  • These fusion proteins serve as readily targetable “DNA scissors” for gene editing applications that enable to perform targeted genome modifications such as sequence insertion,
    deletion, repair and replacement in living cells.

  • NHEJ uses a variety of enzymes to directly join the DNA ends while the more accurate HDR uses a homologous sequence as a template for regeneration of missing DNA sequences
    at the break point.

  • [15][16] Background Genetic engineering as method of introducing new genetic elements into organisms has been around since the 1970s.

  • [32] The method generally adopted for this involves associating two DNA binding proteins – each containing 3 to 6 specifically chosen zinc fingers – with the catalytic domain
    of the FokI endonuclease which need to dimerize to cleave the double-strand DNA.

  • Meganucleases[edit] Meganucleases, discovered in the late 1980s, are enzymes in the endonuclease family which are characterized by their capacity to recognize and cut large
    DNA sequences (from 14 to 40 base pairs).

  • Gene targeting[edit] Homologous recombination[edit] Early methods to target genes to certain sites within a genome of an organism (called gene targeting) relied on homologous

  • This portion could then be linked to sequence recognizing peptides that could lead to very high specificity.

  • [18] Process Double strand break repair[edit] A common form of Genome editing relies on the concept of DNA double stranded break (DSB) repair mechanics.

  • The recognized sequences are short, made up of around 3 base pairs, but by combining 6 to 8 zinc fingers whose recognition sites have been characterized, it is possible to
    obtain specific proteins for sequences of around 20 base pairs.

  • While HDR based gene editing is similar to the homologous recombination based gene targeting, the rate of recombination is increased by at least three orders of magnitude.

  • It has also been possible to knock in genes or alter gene expression patterns.

  • [33] Several approaches are used to design specific zinc finger nucleases for the chosen sequences.

  • It is therefore possible to control the expression of a specific gene.

  • [30] The first step to this was to find an endonuclease whose DNA recognition site and cleaving site were separate from each other, a situation that is not the most common
    among restriction enzymes.

  • TALENs are artificial restriction enzymes designed by fusing the DNA cutting domain of a nuclease to TALE domains, which can be tailored to specifically recognize a unique
    DNA sequence.

  • One major advantage that CRISPR has over the ZFN and TALEN methods is that it can be directed to target different DNA sequences using its ~80nt CRISPR sgRNAs, while both ZFN
    and TALEN methods required construction and testing of the proteins created for targeting each DNA sequence.

  • For instance, the field of synthetic biology which aims to engineer cells and organisms to perform novel functions, is likely to benefit from the ability of engineered nuclease
    to add or remove genomic elements and therefore create complex systems.

  • Using global transcriptomics data to guide experimentation, the CRISPR based genome editing tool has made it feasible to disrupt or remove key genes in order to elucidate
    function in a human setting.

  • CRISPR[edit] Main article: CRISPR gene editing CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats) are genetic elements that bacteria use as a kind of acquired
    immunity to protect against viruses.

  • [56] A potentially successful example of the application of genome editing techniques in crop improvement can be found in banana, where scientists used CRISPR/Cas9 editing
    to inactivate the endogenous banana streak virus in the B genome of banana to overcome a major challenge in banana breeding.

  • Listed below are some specific tasks this method can carry out: • Targeted gene mutation • Gene therapy • Creating chromosome rearrangement • Study gene function with stem
    cells • Transgenic animals • Endogenous gene labeling • Targeted transgene addition Targeted gene modification in animals[edit] The combination of recent discoveries in genetic engineering, particularly gene editing and the latest improvement
    in bovine reproduction technologies (e.g.

  • The growth hormone-regulating gene in the Atlantic salmon is replaced with the growth hormone-regulating gene from the Pacific Chinook salmon and a promoter sequence from
    the ocean pout[50] Thanks to the parallel development of single-cell transcriptomics, genome editing and new stem cell models we are now entering a scientifically exciting period where functional genetics is no longer restricted to animal
    models but can be performed directly in human samples.

  • For instance, site-specific gene addition in major crop species can be used for ‘trait stacking’ whereby several desired traits are physically linked to ensure their co-segregation
    during the breeding processes.

  • [46] MAGE experiments can be divided into three classes, characterized by varying degrees of scale and complexity: (i) many target sites, single genetic mutations; (ii) single
    target site, many genetic mutations; and (iii) many target sites, many genetic mutations.

  • [51] Targeted gene modification in plants[edit] Overview of GEEN workflow and editing possibilities Genome editing using Meganuclease,[52] ZFNs, and TALEN provides a new strategy
    for genetic manipulation in plants and are likely to assist in the engineering of desired plant traits by modifying endogenous genes.

  • Genome editing with engineered nucleases will likely contribute to many fields of life sciences from studying gene functions in plants and animals to gene therapy in humans.

  • It achieves such efficiency because the DNA-binding element consists of an array of TALE subunits, each of them having the capability of recognizing a specific DNA nucleotide
    chain independent from others, resulting in a higher number of target sites with high precision.

  • [9] CRISPR also requires the least amount of expertise in molecular biology as the design lays in the guide RNA instead of the proteins.

  • It is only appropriate for precise editing requiring single nucleotide changes and has found to be highly efficient for this type of editing.

  • New TALE nucleases take about one week and a few hundred dollars to create, with specific expertise in molecular biology and protein engineering.

  • Applications As of 2012 efficient genome editing had been developed for a wide range of experimental systems ranging from plants to animals, often beyond clinical interest,
    and was becoming a standard experimental strategy in research labs.

  • [62] There is a need for reliable design and subsequent test of the nucleases, the absence of toxicity of the nucleases, the appropriate choice of the plant tissue for targeting,
    the routes of induction of enzyme activity, the lack of off-target mutagenesis, and a reliable detection of mutated cases.

  • [58] TALEN fusions have also been used by a U.S. food ingredient company, Calyxt,[59] to improve the quality of soybean oil products[60] and to increase the storage potential
    of potatoes[61] Several optimizations need to be made in order to improve editing plant genomes using ZFN-mediated targeting.

  • In particular CRISPR/Cas9 engineered endonucleases allows the use of multiple guide RNAs for simultaneous Knockouts (KO) in one step by cytoplasmic direct injection (CDI)
    on mammalian zygotes.

  • This is caused by the need of having a specific nucleotide at one end in order to produce the guide RNA that CRISPR uses to repair the double-strand break it induces.

  • Chemically combined, synthetic single-stranded DNA (ssDNA) and a pool of oligionucleotides are introduced at targeted areas of the cell thereby creating genetic modifications.

  • [46] An example of class three was reflected in 2009, where Church and colleagues were able to program Escherichia coli to produce five times the normal amount of lycopene,
    an antioxidant normally found in tomato seeds and linked to anti-cancer properties.

  • Prior to this new revolution, researchers would have to do single-gene manipulations and tweak the genome one little section at a time, observe the phenotype, and start the
    process over with a different single-gene manipulation.

  • Gene editing in fish is currently experimental, but the possibilities include growth, disease resistance, sterility, controlled reproduction, and colour.

  • Cas (CRISPR associated proteins) process these sequences and cut matching viral DNA sequences.

  • [84][85] George Church has compiled a list of potential genetic modifications for possibly advantageous traits such as less need for sleep, cognition-related changes that
    protect against Alzheimer’s disease, disease resistances and enhanced learning abilities along with some of the associated studies and potential negative effects.

  • [84][85] In 2001 Australian researchers Ronald Jackson and Ian Ramshaw were criticized for publishing a paper in the Journal of Virology that explored the potential control
    of mice, a major pest in Australia, by infecting them with an altered mousepox virus that would cause infertility as the provided sensitive information could lead to the manufacture of biological weapons by potential bioterrorists who might
    use the knowledge to create vaccine resistant strains of other pox viruses, such as smallpox, that could affect humans.

  • [83] According to a September 2016 report by the Nuffield Council on Bioethics in the future it may be possible to enhance people with genes from other organisms or wholly
    synthetic genes to for example improve night vision and sense of smell.

  • Gene drive are a potential tool to alter the reproductive rate of invasive species, although there are significant associated risks.

  • The review also found that the risks and benefits of modifying a person’s genome – and having those changes pass on to future generations – are so complex that they demand
    urgent ethical scrutiny.

  • [88] They recommended that clinical trials for genome editing might one day be permitted once answers have been found to safety and efficiency problems “but only for serious
    conditions under stringent oversight.

  • [92] According to a September 2016 report by the Nuffield Council on Bioethics, the simplicity and low cost of tools to edit the genetic code will allow amateurs – or “biohackers”
    – to perform their own experiments, posing a potential risk from the release of genetically modified bugs.

  • Genome editing occurs also as a natural process without artificial genetic engineering.

  • [68] In February 2019, medical scientists working with Sangamo Therapeutics, headquartered in Richmond, California, announced the first ever “in body” human gene editing therapy
    to permanently alter DNA – in a patient with Hunter syndrome.

  • “[89] Risks In the 2016 Worldwide Threat Assessment of the US Intelligence Community statement United States Director of National Intelligence, James R. Clapper, named genome
    editing as a potential weapon of mass destruction, stating that genome editing conducted by countries with regulatory or ethical standards “different from Western countries” probably increases the risk of the creation of harmful biological
    agents or products.

  • CRISPR can help bridge the gap between this model and human clinical trials by creating transgenic disease models in larger animals such as pigs, dogs, and non-human primates.

  • This is advantageous over a virally delivered gene as there is no need to include the full coding sequences and regulatory sequences when only a small proportions of the gene
    needs to be altered as is often the case.

  • [64][65] The expression of the partially replaced genes is also more consistent with normal cell biology than full genes that are carried by viral vectors.

  • Although GEEN has higher efficiency than many other methods in reverse genetics, it is still not highly efficient; in many cases less than half of the treated populations
    obtain the desired changes.

  • [79] Human enhancement[edit] Many transhumanists see genome editing as a potential tool for human enhancement.

  • [44] In November 2018, He Jiankui announced that he had edited two human embryos, to attempt to disable the gene for CCR5, which codes for a receptor that HIV uses to enter

  • [66][67] Extensive research has been done in cells and animals using CRISPR-Cas9 to attempt to correct genetic mutations which cause genetic diseases such as Down syndrome,
    spina bifida, anencephaly, and Turner and Klinefelter syndromes.

  • In the future, a possible method to identify secondary targets would be to capture broken ends from cells expressing the ZFNs and to sequence the flanking DNA using high-throughput

  • [65] Because of the ease of use and cost-efficiency of CRISPR, extensive research is currently being done on it.

  • Such modifications might have unintended consequences which could harm not only the child, but also their future children, as the altered gene would be in their sperm or eggs.

  • [90][91][92] For instance technologies such as CRISPR could be used to make “killer mosquitoes” that cause plagues that wipe out staple crops.


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