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Gene knockout

A gene knockout (abbreviation: KO) is a genetic technique in which one of an organism's genes is made inoperative ("knocked out"
of the organism). However, KO can also refer to the gene that is knocked out or the organism that carries the gene knockout.
Knockout organisms or simply knockouts are used to study gene function, usually by investigating the effect of gene loss.
Researchers draw inferences from the difference between the knockout organism and normal individuals.

The KO technique is essentially the opposite of a gene knockin. 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 heterozygous and homozygousKOs. In the
former, only one of two gene copies (alleles) is knocked out, in the latter both are knocked out.

Contents
Methods
Homologous recombination
Site-specific nucleases
Zinc-fingers
TALENS
CRISPR/Cas9
Knockin
Types
Conditional knockouts
Use
See also
References
External links

Methods
Knockouts are accomplished through a variety of techniques. Originally, naturally occurring mutations were identified and then
gene loss or inactivation had to be established byDNA sequencing or other methods.[1]

Homologous recombination
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.[2] The construct will also contain a minimum of
2kb of homology to the target sequence.[2] The construct can be delivered to stem
cells either through microinjection or electroporation.[2] This method then relies on A laboratory mouse in which a gene
the cell's own repair mechanisms to recombine the DNA construct into the existing affecting hair growth has been
DNA. This results in the sequence of the gene being altered, and most cases the gene knocked out (left), is shown next to a
will be translated into a nonfunctional protein, if it is translated at all. However, this normal lab mouse.
is an inefficient process, as homologous recombination accounts for only 10−2 to 10-3 of DNA integrations.[2][3] Often, the drug
selection marker on the construct is used to select for cells in which the recombination event has occurred.

These stem cells now lacking the gene could be used in vivo, for instance in mice,
by inserting them into early embryos.[2] If the resulting chimeric mouse contained
fspring.[2]
the genetic change in their germline, this could then be passed on of

In diploid organisms, which contain two alleles 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. Wild-type Physcomitrella and
Selective breeding may be required to producehomozygous knockout animals. knockout mosses: Deviating
phenotypes induced in gene-
disruption library transformants.
Site-specific nucleases Physcomitrella wild-type and
transformed plants were grown on
There are currently three methods in use that involve precisely targeting a DNA
minimal Knop medium to induce
sequence in order to introduce a double-stranded break. Once this occurs, the cell's differentiation and development of
repair mechanisms will attempt to repair this double stranded break, often through gametophores. For each plant, an
non-homologous end joining (NHEJ), which involves directly ligating the two cut overview (upper row; scale bar
ends together.[3] This may be done imperfectly, therefore sometimes causing corresponds to 1 mm) and a close-up
insertions or deletions of base pairs, which cause frameshift mutations. These (bottom row; scale bar equals 0.5
mm) are shown. A: Haploid wild-type
mutations can render the gene in which they occur nonfunctional, thus creating a
moss plant completely covered with
knockout of that gene. This process is more efficient than homologous leafy gametophores and close-up of
recombination, and therefore can be more easily used to create biallelic wild-type leaf. B–D: Different
knockouts.[3] mutants.[4]

Zinc-fingers
Zinc-finger nucleases consist of DNA binding domains that can
precisely target a DNA sequence.[3] Each zinc finger can recognize
codons of a desired DNA sequence, and therefore can be modularly
assembled to bind to a particular sequence.[5] These binding
domains are coupled with a restriction endonucleasethat can cause a
double stranded break (DSB) in the DNA.[3] Repair processes may
introduce mutations that destroy functionality of the gene.

TALENS
Transcription activator-like effector nucleases (TALENs) also Fig 1. Frameshift mutation resulting from a single
contain a DNA binding domain and a nuclease that can cleave base pair deletion, causing altered amino acid
DNA.[6] The DNA binding region consists of amino acid repeats sequence and premature stop codon.

that each recognize a single base pair of the desired targeted DNA
sequence.[5] 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.[6]

CRISPR/Cas9
Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 is a method for genome editing that contains a guide RNA
complexed with a Cas9 protein.[5] The guide RNA can be engineered to match a desired DNA sequence through simple
complementary base pairing, as opposed to the time consuming assembly of constructs required by zinc-fingers or TALENs.[7] The
coupled Cas9 will cause a double stranded break in the DNA.[5] Following the same principle as zinc-fingers and TALENs, the
[5]
attempts to repair these double stranded breaks often result in frameshift mutations that result in an nonfunctional gene.
Knockin
Gene knockin is similar to gene knockout, but it replaces a gene with another instead of deleting it.

Types

Conditional knockouts
A conditional knockoutallows gene deletion in a tissue in a time specific manner. This is required in place of a gene knockout if the
null mutation would lead to embryonic death.[8] 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 knock-out. This germ-line can then be crossed to
another germline containing Cre-recombinase which is a viral enzyme that can recognize these sequences, recombines them and
deletes the gene flanked by these sites.

Use
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 genetic
background.

Knockout organisms are also used as screening tools in the development of drugs, to
target specific biological processes or deficiencies by using a specific knockout, or
to understand the mechanism of action of a drug by using a library of knockout
A knockout mouse (left) that is a
organisms spanning the entire genome, such as in Saccharomyces cerevisiae.[9]
model of obesity, compared with a
normal mouse.
See also
Essential gene
Gene knockdown
Conditional gene knockout
Germline
Gene silencing
Planned extinction
Recombineering

References
1. Griffiths AJ, Miller JH, Suzuki DT, Lewontin WC, Gelbart WM (2000).An Introduction to Genetic Analysis(https://ww
w.ncbi.nlm.nih.gov/books/NBK21766/?term=knockout) (7th ed.). New York: W. H. Freeman. ISBN 978-0-7167-3771-
1.
2. Hall, Bradford; Limaye, Advait; Kulkarni, Ashok B. (2009-09-01).Current Protocols in Cell Biology. Current Protocols
in Cell Biology. 44. Wiley-Blackwell. pp. Unit 19.12 19.12.1–17.doi:10.1002/0471143030.cb1912s44(https://doi.org/
10.1002%2F0471143030.cb1912s44). ISBN 978-0471143031. PMC 2782548 (https://www.ncbi.nlm.nih.gov/pmc/arti
cles/PMC2782548). PMID 19731224 (https://www.ncbi.nlm.nih.gov/pubmed/19731224).
3. Santiago, Yolanda; Chan, Edmond; Liu, Pei-Qi; Orlando, Salvatore; Zhang, Lin; Urnov , Fyodor D.; Holmes, Michael
C.; Guschin, Dmitry; Waite, Adam (2008-04-15). "Targeted gene knockout in mammalian cellsby using engineered
zinc-finger nucleases" (http://www.pnas.org/content/105/15/5809). Proceedings of the National Academy of
Sciences. 105 (15): 5809–5814. doi:10.1073/pnas.0800940105(https://doi.org/10.1073%2Fpnas.0800940105) .
ISSN 0027-8424 (https://www.worldcat.org/issn/0027-8424). PMC 2299223 (https://www.ncbi.nlm.nih.gov/pmc/article
s/PMC2299223). PMID 18359850 (https://www.ncbi.nlm.nih.gov/pubmed/18359850).
4. Egener T, Granado J, Guitton M, Hohe A, Holtorf H, Lucht JM, et al. (2002). "High frequency of phenotypic
deviations in Physcomitrella patens plants transformed with a gene-disruption library".
BMC Plant Biology. 2 (1): 6.
doi:10.1186/1471-2229-2-6(https://doi.org/10.1186%2F1471-2229-2-6).
5. Gaj, Thomas; Gersbach, Charles A.; Barbas, Carlos .F(2013). "ZFN, TALEN, and CRISPR/Cas-based methods for
genome engineering" (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3694601). Trends in Biotechnology. 31 (7):
397–405. doi:10.1016/j.tibtech.2013.04.004(https://doi.org/10.1016%2Fj.tibtech.2013.04.004)
. PMC 3694601 (http
s://www.ncbi.nlm.nih.gov/pmc/articles/PMC3694601). PMID 23664777 (https://www.ncbi.nlm.nih.gov/pubmed/23664
777).
6. Joung, J. Keith; Sander, Jeffry D. (January 2013). "TALENs: a widely applicable technology fortargeted genome
editing" (http://www.nature.com/articles/nrm3486). Nature Reviews Molecular Cell Biology. 14 (1): 49–55.
doi:10.1038/nrm3486 (https://doi.org/10.1038%2Fnrm3486). ISSN 1471-0080 (https://www.worldcat.org/issn/1471-0
080). PMC 3547402 (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3547402). PMID 23169466 (https://www.ncbi.nl
m.nih.gov/pubmed/23169466).
7. Ni, Wei; Qiao, Jun; Hu, Shengwei; Zhao, Xinxia; Regouski, Misha; Yang, Min; Polejaeva, Irina A.; Chen, Chuangfu
(2014-09-04). "Efficient Gene Knockout in Goats Using CRISPR/Cas9 System" (https://www.ncbi.nlm.nih.gov/pmc/ar
ticles/PMC4154755). PLOS ONE. 9 (9): e106718. doi:10.1371/journal.pone.0106718(https://doi.org/10.1371%2Fjou
rnal.pone.0106718). ISSN 1932-6203 (https://www.worldcat.org/issn/1932-6203). PMC 4154755 (https://www.ncbi.nl
m.nih.gov/pmc/articles/PMC4154755). PMID 25188313 (https://www.ncbi.nlm.nih.gov/pubmed/25188313).
8. Le, Yunzheng; Sauer, Brian (2001-03-01). "Conditional gene knockout using cre recombin
ase". Molecular
Biotechnology. 17 (3): 269–275. doi:10.1385/MB:17:3:269 (https://doi.org/10.1385%2FMB%3A17%3A3%3A269) .
ISSN 1073-6085 (https://www.worldcat.org/issn/1073-6085). PMID 11434315 (https://www.ncbi.nlm.nih.gov/pubmed/
11434315).
9. "YeastDeletionWebPages" (http://www-sequence.stanford.edu/group/yeast_deletion_project/deletions3.html)
.
Retrieved 21 February 2017.

External links
Diagram of targeted gene replacement
Frontiers in Bioscience Gene Knockout Database (available on archive only)
International Knockout Mouse Consortium
KOMP Repository

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