Gene knockout
Conditional knockouts are particularly useful for studying developmental processes and for understanding the role of a gene in specific cell types or tissues.
Gene knockouts have been widely used in many different organisms, including bacteria, yeast, fruit flies, zebrafish, and mice.
Originally, naturally occurring mutations were identified and then gene loss or inactivation had to be established by DNA sequencing or other methods.
An early instance of the use of this technique in Escherichia coli was published in 1989 by Hamilton, et al.[2] In this experiment, two sequential recombinations were used to delete the gene.
Knockout mice are commonly used to study genes with human equivalents that may have significance for disease.
[4] Homologous recombination is the exchange of genes between two DNA strands that include extensive regions of base sequences that are identical to one another.
In eukaryotic species, bacteria, and some viruses, homologous recombination happens spontaneously and is a useful tool in genetic engineering.
[citation needed] Mario Capecchi, Sir Martin J. Evans, and Oliver Smithies performed groundbreaking research on homologous recombination in mouse stem cells, and they shared the 2007 Nobel Prize in Physiology or Medicine for their findings.
These stem cells now lacking the gene could be used in vivo, for instance in mice, by inserting them into early embryos.
There are currently three methods in use that involve precisely targeting a DNA sequence in order to introduce a double-stranded break.
[9] These binding domains are coupled with a restriction endonuclease that can cause a double stranded break (DSB) in the DNA.
[10] CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a genetic engineering technique that allows for precise editing of the genome.
[citation needed] The process of gene knockout with CRISPR involves three main steps: designing a guide RNA (gRNA) that targets a specific location in the genome, delivering the gRNA and a Cas9 enzyme (which acts as a molecular scissors) to the target cell, and then allowing the cell to repair the cut in the DNA.
This technique can be used in a variety of organisms, including bacteria, yeast, plants, and animals, and it allows scientists to study the function of specific genes by observing the effects of their absence.
CRISPR-based gene knockout is a powerful tool for understanding the genetic basis of disease and for developing new therapies.
It is important to note that CRISPR-based gene knockout, like any genetic engineering technique, has the potential to produce unintended or harmful effects on the organism, so it should be used with caution.
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.
