Triple 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 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 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

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.

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. The construct will also contain a minimum of 2kb of homology to the target sequence. The construct can be delivered to stem cells 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 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. 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. If the resulting chimeric mouse contained the genetic change in their germline, this could then be passed on offspring.

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

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. 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.

Zinc-fingers

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

TALENS

Transcription activator-like effector nucleases (TALENs) also contain a DNA binding domain and a nuclease that can cleave DNA. The DNA binding region consists of amino acid repeats that each recognize a single base pair of the desired targeted DNA sequence. 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.

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. 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. The coupled Cas9 will cause a double stranded break in the DNA. 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.

Knockin

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

Types

Conditional knockouts

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, 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 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 organisms spanning the entire genome, such as in ''Saccharomyces cerevisiae''.

See also

*Essential gene
*Gene knockdown
*Conditional gene knockout
*Germline
*Gene silencing
* Planned extinction
*Recombineering
*Myostatin
*Belgian Blue

References

External links

Diagram of targeted gene replacement
* ttps://web.archive.org/web/20131015201902/http://www.knockoutmouse.org/ International Knockout Mouse Consortiumbr>KOMP Repository

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Genetically modified organisms
Molecular biology techniques
Molecular genetics
Laboratory techniques
Gene expression
Biotechnology