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= Gene Knockout =

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 an 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.

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 far 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
Clustered regularly interspaced short palindromic repeats (CRISPR) 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.

Conditional Knockouts
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. 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.

Mono-Allelic vs. Biallelic Knockout
In diploid organisms, which contain two alleles for most genes, knockouts can be homozygous or heterozygous, also known as monoallelic or biallelic. In the former, only one of two gene copies (alleles) is knocked out, in the latter both are knocked out. Monoallelic knockouts may not completely remove functionality of the gene, as one functional copy remains. Traditional homologous recombination techniques often solely resulted in mono-allelic gene knockouts due to the inefficiency rate. More efficient and precise methods such as zinc-fingers, TALENs, and CRISPR systems can be used to generate biallelic knockouts.

Multiple Gene Knockouts
Several related genes may play a role in the trait under study, in which case multiple knockouts may be performed. These can be referred to as double knockouts, triple knockouts, etc. depending on the number of genes knocked out. These combination knockouts can also be used to see the interactions between different genes by

Deleting:
The directed creation of a KO begins in the test tube with a plasmid, a bacterial artificial chromosome or other DNA construct, and proceeding to cell culture. Individual cells are genetically transfected with the DNA construct. Often the goal is to create a transgenic animal that has the altered gene. If so, embryonic stem cells are genetically transformed and inserted into early embryos. Resulting animals with the genetic change in their germline cells can then often pass the gene knockout to future generations.

The construct is engineered to recombine with the target gene, which is accomplished by incorporating sequences from the gene itself into the construct. Recombination then occurs in the region of that sequence within the gene, resulting in the insertion of a foreign sequence to disrupt the gene. With its sequence interrupted, the altered gene in most cases will be translated into a nonfunctional protein, if it is translated at all.

Because the desired type of DNA recombination is a rare event in the case of most cells and most constructs, the foreign sequence chosen for insertion usually includes a reporter. This enables easy selection of cells or individuals in which knockout was successful. Sometimes the DNA construct inserts into a chromosome without the desired homologous recombination with the target gene. To eliminate such cells, the DNA construct often contains a second region of DNA that allows such cells to be identified and discarded.

To create knockout moss, transfection of protoplasts is the preferred method. Such transformed Physcomitrella-protoplasts directly regenerate into fertile moss plants. Eight weeks after transfection, the plants can be screened for gene targeting via PCR.