Gene 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.
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 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. The drug selection marker on the construct is often used to select cells in which the recombination event has occurred.
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.
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 .
A. 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.
B. 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 .
C. 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 .
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