Editing Gene knockout

{{Short description|Genetic technique}}
{{redirect|Triple knockout|the playoff tournament|Tournament#Knockout tournaments}}
'''Gene knockouts''' (also known as '''gene deletion''' or '''gene inactivation''') are a widely used genetic engineering technique that involves the [[gene targeting|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 gene editing|CRISPR-Cas9]], and [[transcription activator-like effector nuclease|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 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 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 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, 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 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 [[Zygosity|heterozygous and homozygous]] KOs. In the former, only one of two gene copies ([[allele]]s) is knocked out, in the latter both are knocked out.

==Methods==
Knockouts are accomplished through a variety of techniques. Originally, '''naturally occurring [[mutation]]s''' were identified and then gene loss or inactivation had to be established by [[DNA sequencing]] or other methods.<ref>{{cite book | url=https://www.ncbi.nlm.nih.gov/books/NBK21766/?term=knockout | title = An Introduction to Genetic Analysis | edition = 7th | vauthors = Griffiths AJ, Miller JH, Suzuki DT, Lewontin WC, Gelbart WM | isbn = 978-0-7167-3771-1 | location = New York | publisher = W. H. Freeman | year = 2000 }}</ref>[[File:Knockout Mice5006-300.jpg|thumb|A laboratory mouse in which a gene affecting hair growth has been knocked out (left), is shown next to a normal lab mouse.]]

==Gene knockout by mutation==
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.<ref name= Hamilton >{{cite journal |date=September 1989 |vauthors=Hamilton CM, Aldea M, Washburn BK, Babitzke P, Kushner SR |title=New method for generating deletions and gene replacements in ''Escherichia coli'' |journal=Journal of Bacteriology |volume=171 |issue=9 |pages=4617–4622 |doi=10.1128/jb.171.9.4617-4622.1989 |doi-access=free|pmid=2548993 |pmc=210259 }}</ref> In this experiment, two sequential recombinations were used to delete 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 [[XIRP2|Xirp]] proteins in Sudden Unexplained Nocturnal Death Syndrome (SUNDS) and Brugada Syndrome in the Chinese Han Population.<ref name= Huang >{{cite journal |last=Huang |first=Lei |display-authors=etal |date=January 2018 |title=Critical Roles of Xirp Proteins in Cardiac Conduction and Their Rare Variants Identified in Sudden Unexplained Nocturnal Death Syndrome and Brugada Syndrome in Chinese Han Population |journal=J. Am. Heart Assoc. |volume=7 |issue=1 |doi=10.1161/JAHA.117.006320 |doi-access=free|pmid=29306897 |pmc=5778954 }}</ref>

==Gene silencing==
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).<ref>{{Cite journal |last=Caplen |first=Natasha J |date=July 2003 |title=RNAi as a gene therapy approach |url=http://www.tandfonline.com/doi/full/10.1517/14712598.3.4.575 |journal=Expert Opinion on Biological Therapy |language=en |volume=3 |issue=4 |pages=575–586 |doi=10.1517/14712598.3.4.575 |pmid=12831363 |issn=1471-2598}}</ref>

===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.

===Site-specific nucleases===
[[File:Frameshift mutations (13080927393).jpg|thumb|303x303px|Frameshift mutation resulting from a single base pair deletion, causing altered amino acid sequence and premature stop codon]]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 the two cut ends together.<ref name=":1">{{Cite journal|last1=Santiago|first1=Yolanda|last2=Chan|first2=Edmond|last3=Liu|first3=Pei-Qi|last4=Orlando|first4=Salvatore|last5=Zhang|first5=Lin|last6=Urnov|first6=Fyodor D.|last7=Holmes|first7=Michael C.|last8=Guschin|first8=Dmitry|last9=Waite|first9=Adam|date=2008-04-15|title=Targeted gene knockout in mammalian cells by using engineered zinc-finger nucleases|journal=Proceedings of the National Academy of Sciences|volume=105|issue=15|pages=5809–5814|doi=10.1073/pnas.0800940105|issn=0027-8424|pmid=18359850|pmc=2299223|doi-access=free}}</ref> This may be done imperfectly, therefore sometimes causing insertions or deletions of base pairs, which cause [[frameshift mutation]]s. 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.<ref name=":1" />

====Zinc-fingers====
{{Main|Zinc-finger nuclease}}
Zinc-finger nucleases consist of DNA binding domains that can precisely target a DNA sequence.<ref name=":1" /> Each zinc-finger can recognize codons of a desired DNA sequence, and therefore can be modularly assembled to bind to a particular sequence.<ref name=":2" /> These binding domains are coupled with a [[Restriction enzyme|restriction endonuclease]] that can cause a double stranded break (DSB) in the DNA.<ref name=":1" /> Repair processes may introduce mutations that destroy functionality of the gene.{{cn|date=December 2023}}

====TALENS====
{{Main|Transcription activator-like effector nuclease}}
Transcription activator-like effector nucleases ([[TALENs]]) also contain a DNA binding domain and a nuclease that can cleave DNA.<ref name=":3">{{Cite journal|last1=Joung|first1=J. Keith|last2=Sander|first2=Jeffry D.|date=January 2013|title=TALENs: a widely applicable technology for targeted genome editing|journal=Nature Reviews Molecular Cell Biology|volume=14|issue=1|pages=49–55|doi=10.1038/nrm3486|pmid=23169466|issn=1471-0080|pmc=3547402}}</ref> The DNA binding region consists of amino acid repeats that each recognize a single base pair of the desired targeted DNA sequence.<ref name=":2">{{Cite journal|last1=Gaj|first1=Thomas|last2=Gersbach|first2=Charles A.|last3=Barbas|first3=Carlos F.|title=ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering|journal=Trends in Biotechnology|volume=31|issue=7|pages=397–405|doi=10.1016/j.tibtech.2013.04.004|pmid=23664777|pmc=3694601|year=2013}}</ref> 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.<ref name=":3" />

====CRISPR/Cas9====
{{Main|CRISPR}}
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) 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.{{cn|date=December 2023}}

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 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 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.<ref name=":2" /><ref>{{Cite journal|last1=Ni|first1=Wei|last2=Qiao|first2=Jun|last3=Hu|first3=Shengwei|last4=Zhao|first4=Xinxia|last5=Regouski|first5=Misha|last6=Yang|first6=Min|last7=Polejaeva|first7=Irina A.|last8=Chen|first8=Chuangfu|date=2014-09-04|title=Efficient Gene Knockout in Goats Using CRISPR/Cas9 System|journal=PLOS ONE|volume=9|issue=9|pages=e106718|doi=10.1371/journal.pone.0106718|pmid=25188313|pmc=4154755|bibcode=2014PLoSO...9j6718N|issn=1932-6203|doi-access=free}}</ref> The coupled Cas9 will cause a double stranded break in the DNA.<ref name=":2" /> 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.<ref name=":2" /> 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.<ref>{{Cite web |date=2023-06-21 |title=First-of-its-kind noninvasive CRISPR method knocks out anxiety gene |url=https://newatlas.com/medical/intranasal-crispr-gene-editing-reduces-anxiety-in-mice/ |access-date=2024-01-18 |website=New Atlas |language=en-US}}</ref>

===Knock-in===
{{Main|Gene knock-in}}
Gene knock-in is similar to gene knockout, but it replaces a gene with another instead of deleting it.{{cn|date=December 2023}}

==Types==

===Conditional knockouts===
{{Main|Conditional gene knockout}}
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]],<ref>{{Cite journal|last1=Le|first1=Yunzheng|last2=Sauer|first2=Brian|date=2001-03-01|title=Conditional gene knockout using cre recombinase|journal=Molecular Biotechnology|volume=17|issue=3|pages=269–275|doi=10.1385/MB:17:3:269|pmid=11434315|s2cid=41578035|issn=1073-6085}}</ref> or a specific tissue or cell type is of specific interest. This is done by introducing short sequences called loxP sites around 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|Cre-recombinase]] which is a viral enzyme that can recognize these sequences, recombines them and deletes the gene flanked by these sites.<ref>{{Cite journal |last=Kos |first=Claudine H. |date=June 2004 |title=Methods in Nutrition Science: Cre/loxP System for Generating Tissue-specific Knockout Mouse Models |url=https://academic.oup.com/nutritionreviews/article-lookup/doi/10.1111/j.1753-4887.2004.tb00046.x |journal=Nutrition Reviews |language=en |volume=62 |issue=6 |pages=243–246 |doi=10.1111/j.1753-4887.2004.tb00046.x}}</ref> Other recombinases have since been created and employed in conditional knockout experiments.<ref>{{Cite journal |last1=Tian |first1=Xueying |last2=Zhou |first2=Bin |date=January 2021 |title=Strategies for site-specific recombination with high efficiency and precise spatiotemporal resolution |journal=Journal of Biological Chemistry |volume=296 |pages=100509 |doi=10.1016/j.jbc.2021.100509 |doi-access=free |issn=0021-9258 |pmc=8050033 |pmid=33676891}}</ref>

==Use==
[[File:Knockoutmouse80-72.jpg|thumb|A [[knockout mouse]] (left) that is a model of obesity, compared with a normal mouse]]
Knockouts are primarily used to understand the role of a specific [[gene]] or [[DNA]] region by comparing the knockout [[organism]] to a [[wildtype]] with a similar [[Heredity|genetic]] background.{{cn|date=December 2023}}

Knockout [[organisms]] are also used as [[Screening (medicine)|screening]] tools in the development of [[drugs]], to target specific [[biological processes]] or [[Deficiency (medicine)|deficiencies]] by using a specific knockout, or to understand the [[mechanism of action]] of a [[drug]] by using a [[library]] of knockout [[organisms]] spanning the entire [[genome]], such as in ''[[Saccharomyces cerevisiae]]''.<ref>{{cite web|url=http://www-sequence.stanford.edu/group/yeast_deletion_project/deletions3.html|title=YeastDeletionWebPages|access-date=21 February 2017|archive-date=29 September 2012|archive-url=https://web.archive.org/web/20120929010716/http://www-sequence.stanford.edu/group/yeast_deletion_project/deletions3.html|url-status=dead}}</ref>

==See also==
* [[Essential gene]]
* [[Gene knockdown]]
* [[Conditional gene knockout]]
* [[Germline]]
* [[Gene silencing]]
* [[Genome editing]]
* [[Planned extinction]]
* [[Recombineering]]
* [[Myostatin]]
* [[Belgian Blue]]

==References==
{{reflist|refs =

<ref name = "Egener_2002">{{cite journal | title = High frequency of phenotypic deviations in ''Physcomitrella patens'' plants transformed with a gene-disruption library | last1=Egener|first1=Tanja|last2=Granado|first2=José|last3=Guitton|first3=Marie-Christine|last4=Hohe|first4=Annette|last5=Holtorf|first5=Hauke|last6=Lucht|first6=Jan M|last7=Rensing|first7=Stefan A|last8=Schlink|first8=Katja|last9=Schulte|first9=Julia|last10=Schween|first10=Gabriele|last11=Zimmermann|first11=Susanne|last12=Duwenig|first12=Elke|last13=Rak|first13=Bodo|last14=Reski|first14=Ralf | name-list-style = vanc | display-authors = 6 |journal=BMC Plant Biology | date = 2002 | volume = 2 | issue = 1 | pages = 6 | doi = 10.1186/1471-2229-2-6 | pmid=12123528|pmc=117800| doi-access=free| bibcode=2002BMCPB...2....6E}}</ref>

}}

==External links==
* [https://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=iga.figgrp.2286 Diagram of targeted gene replacement]
* [https://web.archive.org/web/20111026040816/http://www.bioscience.org/knockout/knochome.htm Frontiers in Bioscience Gene Knockout Database (available on archive only)]
* [https://web.archive.org/web/20131015201902/http://www.knockoutmouse.org/ International Knockout Mouse Consortium]
* [https://www.komp.org KOMP Repository]
* [https://pubmed.ncbi.nlm.nih.gov/2548993/]
* [https://pubmed.ncbi.nlm.nih.gov/27798100/]
{{genetic engineering}}

{{DEFAULTSORT:Gene Knockout}}
[[Category:Genetically modified organisms]]
[[Category:Molecular biology techniques]]
[[Category:Molecular genetics]]
[[Category:Laboratory techniques]]
[[Category:Gene expression]]
[[Category:Biotechnology]]