Editing Gene knockout (section)

===Homologous recombination===
{{Main|Homologous recombination}}
Homologous recombination 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 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 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 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.{{cn|date=December 2023}} Mario Capecchi, Sir Martin J. Evans, and Oliver Smithies performed groundbreaking research on homologous recombination in mouse stem cells, and they shared the 2007 Nobel Prize in Physiology or Medicine for their findings.<ref name="Nobel 2007">{{cite web|url=http://nobelprize.org/nobel_prizes/medicine/laureates/2007/index.html|title=The Nobel Prize in Physiology or Medicine 2007|publisher=The Nobel Foundation|access-date=December 15, 2008}}</ref> 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.<ref name= Hall>{{Cite journal|last1=Hall|first1=Bradford|last2=Limaye|first2=Advait|last3=Kulkarni|first3=Ashok B.|date=2009-09-01|publisher=Wiley-Blackwell|volume=44|pages=Unit 19.12 19.12.1–17|doi=10.1002/0471143030.cb1912s44|pmid = 19731224|pmc=2782548|isbn = 978-0471143031|title=Overview: Generation of Gene Knockout Mice|journal=Current Protocols in Cell Biology}}</ref> The construct will also contain a minimum of 2kb of [[Sequence homology|homology]] to the target sequence. The construct can be delivered to [[stem cell]]s 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 [[Translation (genetics)|translated]] into a nonfunctional [[protein]], if it is translated at all. However, this is an inefficient process, as homologous recombination accounts for only 10<sup>−2</sup> to 10<sup>−3</sup> of DNA integrations.<ref name= Hall /><ref name=":1" /> Often, the drug selection marker on the construct is used to select for cells in which the recombination event has occurred.

[[Image:Physcomitrella knockout mutants.JPG|thumb|Wild-type [[Physcomitrella patens|''Physcomitrella'']] and [[knockout moss]]es: Deviating [[phenotype]]s induced in gene-disruption library transformants. ''Physcomitrella'' wild-type and transformed plants were grown on minimal Knop medium to induce differentiation and development of [[gametophore]]s. For each plant, an overview (upper row; scale bar corresponds to 1 mm) and a close-up (bottom row; scale bar equals 0.5 mm) are shown. A: Haploid wild-type moss plant completely covered with leafy gametophores and close-up of wild-type leaf. B–E: Different mutants.<ref name = "Egener_2002" />]]
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.<ref name= Hall />

In [[diploid]] organisms, which contain two [[allele]]s for most genes, and may as well contain several related genes that collaborate in 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.