Editing Genetic engineering (section)

===Inserting DNA into the host genome===
{{Main|Gene delivery}}
[[File:Genegun.jpg|left|thumb|upright|A gene gun uses [[biolistics]] to insert DNA into plant tissue.]]
There are a number of techniques used to insert genetic material into the host genome. Some bacteria can naturally [[Competence (biology)|take up foreign DNA]]. This ability can be induced in other bacteria via stress (e.g. [[heat shock|thermal]] or electric shock), which increases the cell membrane's permeability to DNA; up-taken DNA can either integrate with the genome or exist as [[extrachromosomal DNA]]. DNA is generally inserted into animal cells using [[microinjection]], where it can be injected through the cell's [[nuclear envelope]] directly into the [[Cell nucleus|nucleus]], or through the use of [[viral vectors]].<ref>{{cite journal | vauthors = Chen I, Dubnau D | title = DNA uptake during bacterial transformation | journal = Nature Reviews. Microbiology | volume = 2 | issue = 3 | pages = 241–9 | date = March 2004 | pmid = 15083159 | doi = 10.1038/nrmicro844 | s2cid = 205499369 }}</ref>

Plant genomes can be engineered by physical methods or by use of ''[[Agrobacterium]]'' for the delivery of sequences hosted in [[Transfer DNA binary system|T-DNA binary vectors]]. In plants the DNA is often inserted using [[Agrobacterium#Uses in biotechnology|''Agrobacterium''-mediated transformation]],<ref>{{Cite book|url=https://www.ncbi.nlm.nih.gov/books/NBK215771/|title=Methods and Mechanisms for Genetic Manipulation of Plants, Animals, and Microorganisms| author =National Research Council (US) Committee on Identifying and Assessing Unintended Effects of Genetically Engineered Foods on Human Health |date=2004-01-01|publisher=National Academies Press (US)|language=en}}</ref> taking advantage of the ''Agrobacterium''s [[Transfer DNA|T-DNA]] sequence that allows natural insertion of genetic material into plant cells.<ref>{{cite journal | vauthors = Gelvin SB | title = Agrobacterium-mediated plant transformation: the biology behind the "gene-jockeying" tool | journal = Microbiology and Molecular Biology Reviews | volume = 67 | issue = 1 | pages = 16–37, table of contents | date = March 2003 | pmid = 12626681 | pmc = 150518 | doi = 10.1128/MMBR.67.1.16-37.2003 }}</ref> Other methods include [[biolistics]], where particles of gold or tungsten are coated with DNA and then shot into young plant cells,<ref>{{cite book |first1=Graham |last1=Head |last2=Hull |first2=Roger H |last3=Tzotzos |first3=George T. | name-list-style = vanc |title=Genetically Modified Plants: Assessing Safety and Managing Risk |url=https://archive.org/details/geneticallymodif00hull |url-access=limited |publisher=Academic Pr |location=London |year=2009 |page=[https://archive.org/details/geneticallymodif00hull/page/n254 244] |isbn=978-0-12-374106-6 }}</ref> and [[electroporation]], which involves using an electric shock to make the cell membrane permeable to plasmid DNA.

As only a single cell is transformed with genetic material, the organism must be [[Regeneration (biology)|regenerated]] from that single cell. In plants this is accomplished through the use of [[Plant tissue culture|tissue culture]].<ref>{{cite journal | vauthors = Tuomela M, Stanescu I, Krohn K | title = Validation overview of bio-analytical methods | journal = Gene Therapy | volume = 12 Suppl 1 | issue = S1 | pages = S131-8 | date = October 2005 | pmid = 16231045 | doi = 10.1038/sj.gt.3302627 | s2cid = 23000818 | doi-access =  }}</ref><ref>{{Cite book|url=https://books.google.com/books?id=-M4lR-pxqJMC|title=Plant Cell and Tissue Culture|last=Narayanaswamy|first=S.|date=1994|publisher=Tata McGraw-Hill Education|isbn=978-0-07-460277-5|page=vi|language=en}}</ref> In animals it is necessary to ensure that the inserted DNA is present in the [[embryonic stem cells]].<ref>{{Cite book|url=https://www.ncbi.nlm.nih.gov/books/NBK215771/|title=Methods and Mechanisms for Genetic Manipulation of Plants, Animals, and Microorganisms| author =National Research Council (US) Committee on Identifying and Assessing Unintended Effects of Genetically Engineered Foods on Human Health |date=2004|publisher=National Academies Press (US)|language=en}}</ref> Bacteria consist of a single cell and reproduce clonally so regeneration is not necessary. [[Selectable markers]] are used to easily differentiate transformed from untransformed cells. These markers are usually present in the transgenic organism, although a number of strategies have been developed that can remove the selectable marker from the mature transgenic plant.<ref>{{cite journal | vauthors = Hohn B, Levy AA, Puchta H | title = Elimination of selection markers from transgenic plants | journal = Current Opinion in Biotechnology | volume = 12 | issue = 2 | pages = 139–43 | date = April 2001 | pmid = 11287227 | doi = 10.1016/S0958-1669(00)00188-9 }}</ref>

[[File:Agrobacterium-tumefaciens.png|thumb|upright|''[[A. tumefaciens]]'' attaching itself to a carrot cell]]

Further testing using PCR, [[Southern hybridization]], and [[DNA sequencing]] is conducted to confirm that an organism contains the new gene.<ref>{{Cite book|url=https://books.google.com/books?id=aGkXFmqOcyIC|title=Genetic Engineering: Principles and Methods|last=Setlow|first=Jane K. | name-list-style = vanc |date=2002-10-31|publisher=Springer Science & Business Media|isbn=978-0-306-47280-0|page=109|language=en}}</ref> These tests can also confirm the chromosomal location and copy number of the inserted gene. The presence of the gene does not guarantee it will be [[Gene expression|expressed]] at appropriate levels in the target tissue so methods that look for and measure the gene products (RNA and protein) are also used. These include [[northern hybridization|northern hybridisation]], quantitative [[RT-PCR]], [[Western blot]], [[immunofluorescence]], [[ELISA]] and phenotypic analysis.<ref>{{cite journal | vauthors = Deepak S, Kottapalli K, Rakwal R, Oros G, Rangappa K, Iwahashi H, Masuo Y, Agrawal G | title = Real-Time PCR: Revolutionizing Detection and Expression Analysis of Genes | journal = Current Genomics | volume = 8 | issue = 4 | pages = 234–51 | date = June 2007 | pmid = 18645596 | pmc = 2430684 | doi = 10.2174/138920207781386960 }}</ref>

The new genetic material can be inserted randomly within the host genome or targeted to a specific location. The technique of [[gene targeting]] uses [[homologous recombination]] to make desired changes to a specific [[endogenous]] gene.  This tends to occur at a relatively low frequency in plants and animals and generally requires the use of [[selectable markers]]. The frequency of gene targeting can be greatly enhanced through [[genome editing]]. Genome editing uses artificially engineered [[nuclease]]s that create specific [[double-strand breaks|double-stranded breaks]] at desired locations in the genome, and use the cell's endogenous mechanisms to repair the induced break by the natural processes of [[homologous recombination]] and [[Nonhomologous end joining|nonhomologous end-joining]]. There are four families of engineered nucleases: [[meganuclease]]s,<ref>{{cite journal | vauthors = Grizot S, Smith J, Daboussi F, Prieto J, Redondo P, Merino N, Villate M, Thomas S, Lemaire L, Montoya G, Blanco FJ, Pâques F, Duchateau P | title = Efficient targeting of a SCID gene by an engineered single-chain homing endonuclease | journal = Nucleic Acids Research | volume = 37 | issue = 16 | pages = 5405–19 | date = September 2009 | pmid = 19584299 | pmc = 2760784 | doi = 10.1093/nar/gkp548 }}</ref><ref>{{cite journal | vauthors = Gao H, Smith J, Yang M, Jones S, Djukanovic V, Nicholson MG, West A, Bidney D, Falco SC, Jantz D, Lyznik LA | title = Heritable targeted mutagenesis in maize using a designed endonuclease | journal = The Plant Journal | volume = 61 | issue = 1 | pages = 176–87 | date = January 2010 | pmid = 19811621 | doi = 10.1111/j.1365-313X.2009.04041.x | doi-access =  }}</ref> [[zinc finger nuclease]]s,<ref>{{cite journal | vauthors = Townsend JA, Wright DA, Winfrey RJ, Fu F, Maeder ML, Joung JK, Voytas DF | title = High-frequency modification of plant genes using engineered zinc-finger nucleases | journal = Nature | volume = 459 | issue = 7245 | pages = 442–5 | date = May 2009 | pmid = 19404258 | pmc = 2743854 | doi = 10.1038/nature07845 | bibcode = 2009Natur.459..442T }}</ref><ref>{{cite journal | vauthors = Shukla VK, Doyon Y, Miller JC, DeKelver RC, Moehle EA, Worden SE, Mitchell JC, Arnold NL, Gopalan S, Meng X, Choi VM, Rock JM, Wu YY, Katibah GE, Zhifang G, McCaskill D, Simpson MA, Blakeslee B, Greenwalt SA, Butler HJ, Hinkley SJ, Zhang L, Rebar EJ, Gregory PD, Urnov FD | title = Precise genome modification in the crop species Zea mays using zinc-finger nucleases | journal = Nature | volume = 459 | issue = 7245 | pages = 437–41 | date = May 2009 | pmid = 19404259 | doi = 10.1038/nature07992 | bibcode = 2009Natur.459..437S | s2cid = 4323298 }}</ref> [[transcription activator-like effector nuclease]]s (TALENs),<ref>{{cite journal | vauthors = Christian M, Cermak T, Doyle EL, Schmidt C, Zhang F, Hummel A, Bogdanove AJ, Voytas DF | title = Targeting DNA double-strand breaks with TAL effector nucleases | journal = Genetics | volume = 186 | issue = 2 | pages = 757–61 | date = October 2010 | pmid = 20660643 | pmc = 2942870 | doi = 10.1534/genetics.110.120717 }}</ref><ref>{{cite journal | vauthors = Li T, Huang S, Jiang WZ, Wright D, Spalding MH, Weeks DP, Yang B | title = TAL nucleases (TALNs): hybrid proteins composed of TAL effectors and FokI DNA-cleavage domain | journal = Nucleic Acids Research | volume = 39 | issue = 1 | pages = 359–72 | date = January 2011 | pmid = 20699274 | pmc = 3017587 | doi = 10.1093/nar/gkq704 }}</ref> and the Cas9-guideRNA system (adapted from [[CRISPR gene editing|CRISPR]]).<ref>{{cite journal | vauthors = Esvelt KM, Wang HH | title = Genome-scale engineering for systems and synthetic biology | journal = Molecular Systems Biology | volume = 9 | page = 641 | year = 2013 | pmid = 23340847 | pmc = 3564264 | doi = 10.1038/msb.2012.66 }}</ref><ref>{{cite book | vauthors = Tan WS, Carlson DF, Walton MW, Fahrenkrug SC, Hackett PB | chapter = Precision editing of large animal genomes | volume = 80 | pages = 37–97 | year = 2012 | pmid = 23084873 | pmc = 3683964 | doi = 10.1016/B978-0-12-404742-6.00002-8 | isbn = 978-0-12-404742-6 | title = Advances in Genetics Volume 80 }}</ref> TALEN and CRISPR are the two most commonly used and each has its own advantages.<ref name="MalzahnLowderQi2017">{{cite journal | vauthors = Malzahn A, Lowder L, Qi Y | title = Plant genome editing with TALEN and CRISPR | journal = Cell & Bioscience | volume = 7 | page = 21 | date = 2017-04-24 | pmid = 28451378 | pmc = 5404292 | doi = 10.1186/s13578-017-0148-4 | doi-access = free }}</ref> TALENs have greater target specificity, while CRISPR is easier to design and more efficient.<ref name="MalzahnLowderQi2017" /> In addition to enhancing gene targeting, engineered nucleases can be used to introduce mutations at endogenous genes that generate a [[gene knockout]].<ref>{{cite journal | vauthors = Ekker SC | title = Zinc finger-based knockout punches for zebrafish genes | journal = Zebrafish | volume = 5 | issue = 2 | pages = 121–3 | year = 2008 | pmid = 18554175 | pmc = 2849655 | doi = 10.1089/zeb.2008.9988 }}</ref><ref>{{cite journal | vauthors = Geurts AM, Cost GJ, Freyvert Y, Zeitler B, Miller JC, Choi VM, Jenkins SS, Wood A, Cui X, Meng X, Vincent A, Lam S, Michalkiewicz M, Schilling R, Foeckler J, Kalloway S, Weiler H, Ménoret S, Anegon I, Davis GD, Zhang L, Rebar EJ, Gregory PD, Urnov FD, Jacob HJ, Buelow R | title = Knockout rats via embryo microinjection of zinc-finger nucleases | journal = Science | volume = 325 | issue = 5939 | page = 433 | date = July 2009 | pmid = 19628861 | pmc = 2831805 | doi = 10.1126/science.1172447 | bibcode = 2009Sci...325..433G }}</ref>