Gene knockout

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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 homozygous KOs. In the former, only one of two gene copies (alleles) is knocked out, in the latter both are knocked out.

Methods[edit]

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

A laboratory mouse in which a gene affecting hair growth has been knocked out (left), is shown next to a normal lab mouse.

Homologous Recombination[edit]

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 an 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 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 translated into a nonfunctional protein, if it is translated at all. However, this 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.

Wild-type Physcomitrella and knockout mosses: Deviating phenotypes induced in gene-disruption library transformants. Physcomitrella wild-type and transformed plants were grown on minimal Knop medium to induce differentiation and development of gametophores. 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–D: Different mutants.[4]

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 the genetic change in their germline, this could then be passed on offspring.[2]

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. Selective breeding may be required to produce homozygous knockout animals.

Site-Specific Nucleases[edit]

Fig 1. 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.[3] This may be done imperfectly, therefore sometimes causing insertions or deletions of base pairs, which cause frameshift mutations. 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.[3]

Zinc-Fingers[edit]

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 endonuclease that can cause a double stranded break (DSB) in the DNA.[3] Repair processes may introduce mutations that destroy functionality of the gene.

TALENS[edit]

Transcription activator-like effector nucleases (TALENs) also contain a DNA binding domain and a nuclease that can cleave DNA.[6] The DNA binding region consists of amino acid repeats 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[edit]

Clustered regularly interspaced short palindromic repeats (CRISPR) 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 attempts to repair these double stranded breaks often result in frameshift mutations that result in an nonfunctional gene.[5]

Knockin[edit]

Gene knockin is similar to gene knockout, but it replaces a gene with another instead of deleting it.

Types[edit]

Conditional Knockouts[edit]

A conditional knockout allows 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[edit]

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 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 organisms spanning the entire genome, such as in Saccharomyces cerevisiae.[9]

See also[edit]

References[edit]

  1. ^ Griffiths AJ, Miller JH, Suzuki DT, Lewontin WC, Gelbart WM (2000). An Introduction to Genetic Analysis (7th ed.). New York: W. H. Freeman. ISBN 978-0-7167-3771-1. 
  2. ^ a b c d e f Hall, Bradford; Limaye, Advait; Kulkarni, Ashok B. (2009-09-01). Overview: Generation of Gene Knockout Mice. 44. Wiley-Blackwell. doi:10.1002/0471143030.cb1912s44. 
  3. ^ a b c d e 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 cells by using engineered zinc-finger nucleases". Proceedings of the National Academy of Sciences. 105 (15): 5809–5814. doi:10.1073/pnas.0800940105. ISSN 0027-8424. PMC 2299223Freely accessible. PMID 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. 
  5. ^ a b c d e Gaj, Thomas; Gersbach, Charles A.; Barbas, Carlos F. "ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering". Trends in Biotechnology. 31 (7): 397–405. doi:10.1016/j.tibtech.2013.04.004. PMC 3694601Freely accessible. 
  6. ^ a b Joung, J. Keith; Sander, Jeffry D. (January 2013). "TALENs: a widely applicable technology for targeted genome editing". Nature Reviews Molecular Cell Biology. 14 (1): 49–55. doi:10.1038/nrm3486. ISSN 1471-0080. PMC 3547402Freely accessible. 
  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". PLOS ONE. 9 (9): e106718. doi:10.1371/journal.pone.0106718. ISSN 1932-6203. 
  8. ^ Le, Yunzheng; Sauer, Brian (2001-03-01). "Conditional gene knockout using cre recombinase". Molecular Biotechnology. 17 (3): 269–275. doi:10.1385/MB:17:3:269. ISSN 1073-6085. 
  9. ^ "YeastDeletionWebPages". Retrieved 21 February 2017. 

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