Editing Genetically modified organism (section)

== Production ==
{{Main|Genetic engineering techniques}}
[[File:Genegun.jpg|thumb|upright|A gene gun uses [[biolistics]] to insert DNA into plant tissue.|alt=]]

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 (biology)|cell]]<ref>{{cite book|url=https://books.google.com/books?id=g1v6WMHVkTgC|title=An Introduction to Genetic Engineering| vauthors = Nicholl DS |date=29 May 2008|publisher=Cambridge University Press|isbn=978-1-139-47178-7|pages=34 }}</ref> or [[Gene synthesis|artificially synthesized]].<ref>{{cite journal | vauthors = Liang J, Luo Y, Zhao H | title = Synthetic biology: putting synthesis into biology | journal = Wiley Interdisciplinary Reviews: Systems Biology and Medicine | volume = 3 | issue = 1 | pages = 7–20 | year = 2011 | pmid = 21064036 | pmc = 3057768 | doi = 10.1002/wsbm.104 }}</ref> If the chosen gene or the donor organism's [[genome]] has been well studied it may already be accessible from a [[Library (biology)|genetic library]]. The gene is then combined with other genetic elements, including a [[Promoter (biology)|promoter]] and [[Terminator (genetics)|terminator]] region and a [[selectable marker]].<ref>{{cite journal | vauthors = Berg P, Mertz JE | title = Personal reflections on the origins and emergence of recombinant DNA technology | journal = Genetics | volume = 184 | issue = 1 | pages = 9–17 | date = January 2010 | pmid = 20061565 | pmc = 2815933 | doi = 10.1534/genetics.109.112144 }}</ref>

A number of techniques are available for [[Gene delivery|inserting the isolated gene into the host genome]]. Bacteria can be induced to take up foreign DNA, usually by exposed [[Heat shock response|heat shock]] or [[electroporation]].<ref>{{cite journal | vauthors = Rahimzadeh M, Sadeghizadeh M, Najafi F, Arab S, Mobasheri H | title = Impact of heat shock step on bacterial transformation efficiency | journal = Molecular Biology Research Communications | volume = 5 | issue = 4 | pages = 257–261 | date = December 2016 | pmid = 28261629 | pmc = 5326489 }}</ref> 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> In plants the DNA is often inserted using [[Agrobacterium#Uses in biotechnology|''Agrobacterium''-mediated recombination]],<ref name="NRC_GMO_Foods">{{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=1 January 2004|publisher=National Academies Press (US)}}</ref><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> [[biolistics]]<ref>{{cite book | vauthors = Head G, Hull RH, Tzotzos GT |title= Genetically Modified Plants: Assessing Safety and Managing Risk |publisher=Academic Press |location=London |year=2009 |page=244 |isbn=978-0-12-374106-6 }}</ref> or electroporation.

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 [[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 | 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| vauthors = Narayanaswamy S |date=1994|publisher=Tata McGraw-Hill Education|isbn=978-0-07-460277-5|pages=vi}}</ref> In animals it is necessary to ensure that the inserted DNA is present in the [[embryonic stem cells]].<ref name="NRC_GMO_Foods" /> Further testing using [[Polymerase chain reaction|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&q=Genetic+Engineering+analysis+of+DNA+PCR+Southern+sequencing|title=Genetic Engineering: Principles and Methods| vauthors = Setlow JK  |date=31 October 2002|publisher=Springer Science & Business Media|isbn=978-0-306-47280-0|pages=109 }}</ref>

Traditionally the new genetic material was inserted randomly within the host genome. [[Gene targeting]] techniques, which creates [[double-strand breaks|double-stranded breaks]] and takes advantage on the cells natural [[homologous recombination]] repair systems, have been developed to target insertion to exact [[Locus (genetics)|locations]]. [[Genome editing]] uses artificially engineered [[nuclease]]s that create breaks at specific points. 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).<ref>{{cite journal | vauthors = Esvelt KM, Wang HH | title = Genome-scale engineering for systems and synthetic biology | journal = Molecular Systems Biology | volume = 9 | pages = 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 | title = Advances in Genetics Volume 80 | 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 }}</ref> TALEN and CRISPR are the two most commonly used and each has its own advantages.<ref name=":5">{{cite journal | vauthors = Malzahn A, Lowder L, Qi Y | title = Plant genome editing with TALEN and CRISPR | journal = Cell & Bioscience | volume = 7 | pages = 21 | date = 24 April 2017 | 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=":5" />